Visualization of the type III secretion sorting platform of Shigella flexneri

Bacterial type III secretion machines are widely used to inject virulence proteins into eukaryotic host cells. These secretion machines are evolutionarily related to bacterial flagella and consist of a large cytoplasmic complex, a transmembrane basal body, and an extracellular needle. The cytoplasmic complex forms a sorting platform essential for effector selection and needle assembly, but it remains largely uncharacterized. Here we use high-throughput cryoelectron tomography (cryo-ET) to visualize intact machines in a virulent Shigella flexneri strain genetically modified to produce minicells capable of interaction with host cells. A high-resolution in situ structure of the intact machine determined by subtomogram averaging reveals the cytoplasmic sorting platform, which consists of a central hub and six spokes, with a pod-like structure at the terminus of each spoke. Molecular modeling of wild-type and mutant machines allowed us to propose a model of the sorting platform in which the hub consists mainly of a hexamer of the Spa47 ATPase, whereas the MxiN protein comprises the spokes and the Spa33 protein forms the pods. Multiple contacts among those components are essential to align the Spa47 ATPase with the central channel of the MxiA protein export gate to form a unique nanomachine. The molecular architecture of the Shigella type III secretion machine and its sorting platform provide the structural foundation for further dissecting the mechanisms underlying type III secretion and pathogenesis and also highlight the major structural distinctions from bacterial flagella.Type III secretion systems (T3SSs) are essential virulence determinants for many Gram-negative pathogens. The injectisome, also known as the needle complex, is the central T3SS machine required to inject effector proteins from the bacterium into eukaryotic host cells (1, 2). The injectisome has three major components: an extracellular needle, a basal body, and a cytoplasmic complex (3). Contact with a host cell membrane triggers activation of the injectisome and the insertion of a translocon pore into the target cell membrane. The entire complex then serves as a conduit for direct translocation of effectors (1, 2). Assembly of a functional T3SS requires recognition and sorting of specific secretion substrates in a well-defined order by the cytoplasmic complex (4, 5). Furthermore, genes encoding the cytoplasmic complex are regulated by physical and environmental signals (6), providing temporal control of the injection of effector proteins and thereby optimizing invasion and virulence.Significant progress has been made in elucidating T3SS structures from many different bacteria (7, 8). 3D reconstructions of purified injectisomes from Salmonella and Shigella, together with the atomic structures of major basal body proteins, have provided a detailed view of basal body architecture (9, 10). Recent in situ structures of injectisomes from Shigella flexneri, Salmonella enterica, and Yersinia enterocolitica revealed an export gate and the structural flexibility of the basal body (11, 12). Unfortunately, these in situ structures from intact bacteria (11, 12) did not reveal any evident densities related to the proposed model of the cytoplasmic complex (8, 13).The flagellar C ring is the cytoplasmic complex in evolutionarily related flagellar systems. It is composed of flagellar proteins FliG, FliM, and FliN and plays an essential role in flagellar assembly, rotation, and switching (14). Large drum-shaped structures of the flagellar C ring have been determined in both purified basal bodies (15, 16) and in situ motors (17–19). Similarly, electron microscopy analysis in Shigella indicated that the Spa33 protein (a homolog of the flagellar proteins FliN and FliM) is localized beneath the basal body via interactions with MxiG and MxiJ and is an essential component of the putative C ring (20). Recent experimental evidence suggests that the putative C ring provides a sorting platform for the recognition and secretion of the substrates in S. enterica (5). This sorting platform consists of three proteins, SpaO, OrgA, and OrgB, which are highly conserved among other T3SSs (21) (SI Appendix, Table S1). Despite its critical roles, little is still known about the structure and assembly of the cytoplasmic sorting platform in T3SS. In this study, we choose S. flexneri as a model system to study the intact T3SS machine and its cytoplasmic complex, mainly because a wealth of structural, biochemical, and functional information is available for the S. flexneri T3SS (22).     

Calcium levels in the Golgi complex regulate clustering and apical sorting of GPI-APs in polarized epithelial cells

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are lipid-associated luminal secretory cargoes selectively sorted to the apical surface of the epithelia where they reside and play diverse vital functions. Cholesterol-dependent clustering of GPI-APs in the Golgi is the key step driving their apical sorting and their further plasma membrane organization and activity; however, the specific machinery involved in this Golgi event is still poorly understood. In this study, we show that the formation of GPI-AP homoclusters (made of single GPI-AP species) in the Golgi relies directly on the levels of calcium within cisternae. We further demonstrate that the TGN calcium/manganese pump, SPCA1, which regulates the calcium concentration within the Golgi, and Cab45, a calcium-binding luminal Golgi resident protein, are essential for the formation of GPI-AP homoclusters in the Golgi and for their subsequent apical sorting. Down-regulation of SPCA1 or Cab45 in polarized epithelial cells impairs the oligomerization of GPI-APs in the Golgi complex and leads to their missorting to the basolateral surface. Overall, our data reveal an unexpected role for calcium in the mechanism of GPI-AP apical sorting in polarized epithelial cells and identify the molecular machinery involved in the clustering of GPI-APs in the Golgi.

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are localized on the apical surface of most epithelia, where they exert their physiological functions, which are regulated by their spatiotemporal compartmentalization.In polarized epithelial cells, the organization of GPI-APs at the apical surface is driven by the mechanism of apical sorting, which relies on the formation of GPI-AP homoclusters in the Golgi apparatus (1, 2). GPI-AP homoclusters (containing a single GPI-AP species) form uniquely in the Golgi apparatus of fully polarized cells (and not in nonpolarized cells) in a cholesterol-dependent manner (1, 3, 4). Once formed, GPI-AP homoclusters become insensitive to cholesterol depletion, suggesting that protein–protein interactions stabilize them (1, 2). At the apical membrane, newly arrived homoclusters coalesce into heteroclusters (containing at least two different GPI-AP species) that are sensitive to cholesterol depletion (1). Of importance, in the absence of homoclustering in the Golgi (e.g., in nonpolarized epithelial cells), GPI-APs remain in the form of monomers and dimers and do not cluster at the cell surface (1, 5). Thus, the organization of GPI-APs at the apical plasma membrane of polarized cells strictly depends on clustering mechanisms in the Golgi apparatus allowing their apical sorting. This is different from what was shown in fibroblasts where clustering of GPI-APs occurs from monomer condensation at the plasma membrane, indicating that distinct mechanisms regulate GPI-AP clustering in polarized epithelial cells and fibroblasts (1, 6, 7). Furthermore, in polarized epithelial cells, the spatial organization of clusters also appears to regulate the biological activity of the proteins (1) so that GPI-APs are fully functional only when properly sorted to the apical surface and less active in the case of missorting to the basolateral domain (1, 8, 9). Understanding the mechanism of GPI-AP apical sorting in the Golgi apparatus is therefore crucial to decipher their organization at the plasma membrane and the regulation of their activity. The determinants for protein apical sorting have been difficult to uncover compared to the ones for basolateral sorting (10–14). Besides a role of cholesterol, the molecular factors regulating the clustering-based mechanism of GPI-AP sorting in polarized epithelial cells are unknown. Here, we analyzed the possible role of the actin cytoskeleton and of calcium levels in the Golgi. The actin cytoskeleton is not only critical for the maintenance of the Golgi structure and its mechanical properties but also provides the structural support favoring carrier biogenesis (15–18). The Golgi exit of various cargoes is altered in cells treated with drugs either depolymerizing or stabilizing actin filaments (19, 20), and the post-Golgi trafficking is affected either by the knockdown of the expression of some actin-binding proteins, which regulate actin dynamics, or by the overexpression of their mutants (12, 21–23), all together revealing the critical role of actin dynamics for protein trafficking. Only few studies have shown the involvement of actin remodeling proteins in polarized trafficking, mostly in selectively mediating the apical and basolateral trafficking of transmembrane proteins [refs. 24–26; and reviewed in ref. 27]; thus, it remains unclear whether actin filaments play a role in protein sorting in polarized cells.On the other hand, the Golgi apparatus exhibits high calcium levels that have been revealed to be essential for protein processing and the sorting of some secreted soluble proteins in nonpolarized cells (28–31). Moreover, a functional interplay between the actin cytoskeleton and Golgi calcium in modulating protein sorting in nonpolarized cells has been shown (22).In this study, we report that in epithelial cells, actin perturbation does not impair GPI-AP clustering capacity in the Golgi and therefore their apical sorting. In contrast, we found that the Golgi organization of GPI-APs is drastically perturbed upon calcium depletion and that the amount of calcium in the Golgi cisternae is critical for the formation of GPI-AP homoclusters. We further show that the TGN calcium/manganese pump, SPCA1 (secretory pathway Ca(2+)-ATPase pump type 1), which controls the Golgi calcium concentration (32), and Cab45, a calcium-binding luminal Golgi resident protein previously described to be involved in the sorting of a subset of soluble cargoes (33, 34), are essential for the formation of GPI-APs homoclusters in the Golgi and for their subsequent apical sorting. Indeed, down-regulation of SPCA1 or Cab45 expression impairs the oligomerization of GPI-APs in the Golgi complex and leads to their missorting to the basolateral surface but does not affect apical or basolateral transmembrane proteins. Overall, our data reveal an unexpected role for calcium in the mechanism of GPI-AP apical sorting in polarized epithelial cells and identify the molecular machinery involved in the clustering of GPI-APs in the Golgi.     

Differential vesicular sorting of AMPA and GABAA receptors

In mature neurons AMPA receptors cluster at excitatory synapses primarily on dendritic spines, whereas GABA_A receptors cluster at inhibitory synapses mainly on the soma and dendritic shafts. The molecular mechanisms underlying the precise sorting of these receptors remain unclear. By directly studying the constitutive exocytic vesicles of AMPA and GABA_A receptors in vitro and in vivo, we demonstrate that they are initially sorted into different vesicles in the Golgi apparatus and inserted into distinct domains of the plasma membrane. These insertions are dependent on distinct Rab GTPases and SNARE complexes. The insertion of AMPA receptors requires SNAP25–syntaxin1A/B–VAMP2 complexes, whereas insertion of GABA_A receptors relies on SNAP23–syntaxin1A/B–VAMP2 complexes. These SNARE complexes affect surface targeting of AMPA or GABA_A receptors and synaptic transmission. Our studies reveal vesicular sorting mechanisms controlling the constitutive exocytosis of AMPA and GABA_A receptors, which are critical for the regulation of excitatory and inhibitory responses in neurons.In the mammalian central nervous system, neurons receive excitatory and inhibitory signals at synapses. Specific receptors at postsynaptic membranes are activated by neurotransmitters released by presynaptic terminals. Most fast excitatory neurotransmission is mediated by AMPA receptors, the majority of which are heterotetramers of GluA1/GluA2 or GluA2/GluA3 subunits in the hippocampus (1). Fast synaptic inhibition is largely mediated by GABA_A receptors, which are predominantly heteropentamers of two α subunits, two β subunits, and one γ or δ subunit in the hippocampus (2). Numerous studies have demonstrated AMPA receptors are selectively localized at excitatory synapses on dendritic spines, whereas GABA_A receptors cluster at inhibitory synapses localized on dendritic shafts and the soma (3). This segregation of excitatory and inhibitory receptors requires highly precise sorting machinery to target receptors to distinct synapses opposing specific presynaptic terminals. However, it is still not clear whether the receptors are sorted before exocytosis into the plasma membrane or are differentially localized only after exocytosis. For example in a “plasma membrane sorting model,” different receptors could be pooled into the same vesicle and inserted along the somatodendritic membrane. The initial sorting would occur on the plasma membrane, where inserted receptors would be segregated by lateral diffusion and stabilization at different postsynaptic zones. Alternatively, in a “vesicle sorting model,” different receptors would first be sorted into different vesicles during intracellular trafficking processes and independently inserted to the plasma membrane, where receptors could be further targeted to specific zones and stabilized by synaptic scaffolds. To date there has been no direct evidence to support either model. However, a large body of literature suggests that the exocytic pathways of AMPA and GABA_A receptors have similar but also distinct properties (1, 2).Increasing evidence has suggested roles for the SNARE protein family in vesicular trafficking of AMPA and GABA_A receptors (4–17). SNAREs are a large family of membrane-associated proteins critical for many intracellular membrane trafficking events. The family is subdivided into v-SNAREs (synaptobrevin/VAMP, vesicle-associated membrane proteins) and t-SNAREs (syntaxins and SNAP25, synaptosomal-associated protein of 25 kDa) based on their localization on trafficking vesicles or target membranes, respectively. To mediate vesicle fusion with target membranes, SNARE proteins form a four-helix bundle (SNARE complex) consisting of two coiled-coil domains from SNAP25, one coiled-coil domain from syntaxin, and a coiled-coil domain from VAMPs (18). Formation of the helical bundle can be disrupted by neurotoxins, which specifically cleave different SNARE proteins (19). Each SNARE subfamily is composed of genes with high homology but different tissue specificity and subcellular localization. It remains to be determined whether individual SNAREs play specific roles in regulating the membrane trafficking of individual proteins.To address how AMPA and GABA_A receptors are sorted in the exocytic pathway and what molecules are involved in regulating exocytosis of these receptors, we specifically studied constitutive exocytosis of AMPA and GABA_A receptor subunits using total internal reflection fluorescence microscopy (TIRFM) in combination with immunocytochemistry, electrophysiology, and electron microscopy methods. Together, we revealed that AMPA and GABA_A receptors are initially sorted into different vesicles in the Golgi apparatus and delivered to different domains at the plasma membrane and are regulated by specific Rab proteins and SNARE complexes. These results reveal fundamental mechanisms underlying the sorting of excitatory and inhibitory neurotransmitter receptors in neurons and uncover the specific trafficking machinery involved in the constitutive exocytosis of each receptor type.     

A SURF4-to-proteoglycan relay mechanism that mediates the sorting and secretion of a tagged variant of sonic hedgehog

Sonic Hedgehog (Shh) is a key signaling molecule that plays important roles in various developmental processes in mammals. Although the signal transduction pathway activated by Shh is well understood, the regulation of its secretion remains unclear. Newly synthesized Shh is imported into the endoplasmic reticulum (ER), where it undergoes a series of posttranslational modifications to produce the mature lipid-modified amino-terminal fragment. Here, we have analyzed the molecular mechanisms that mediate secretion of the N-terminal fragment of Shh (ShhN). We found that the Cardin–Weintraub (CW) motif in Shh is necessary and sufficient for ER-to-Golgi transport of ShhN. Mechanistic analyses revealed that a cargo receptor, Surfeit locus protein 4 (SURF4), interacts directly with the CW motif of ShhN to regulate packaging of ShhN into COPII vesicles. ShhN and SURF4 interact with each other at the ER and separate from each other after entering the Golgi. The CW motif is known to interact with proteoglycans (PGs) that are predominantly synthesized at the Golgi. Interestingly, we found that PGs compete with SURF4 to bind ShhN and that inhibiting synthesis of PGs causes defects in export of ShhN from the trans Golgi network (TGN). SURF4 and PG maturation are also important for intracellular traffic of full length Shh in mammalian cells. Our study suggests a SURF4-to-PG relay mechanism that mediates the sorting and secretion of Shh, providing insight into the biosynthetic trafficking of Shh.

The Hedgehog (Hh) signaling pathway plays an important role in various developmental processes in metazoans (1, 2). Mutations of key components that regulate Hh signaling are associated with many human diseases (3). Hh was first found in the Drosophila larval epidermis. It mediates larval segment development and adult appendage patterning (4). In mammals, there are three Hh-family members, Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Ihh regulates the proliferation and differentiation of chondrocytes (5). Dhh functions in gonads, regulating testis organogenesis, spermatogenesis (6, 7), and follicle development in the ovary (8). Shh functions more extensively than the other two Hh members: it regulates embryonic patterning (4), specification of cell types in the nervous system (9), axon guidance (10), cell differentiation, and organ development (11).Hh is synthesized as a full-length precursor Hh (HhFL). After entering the endoplasmic reticulum (ER), HhFL is autocleaved into two parts: an N-terminal Hedge domain (HhN) and a C-terminal Hog domain (HhC) (1). HhC is degraded through ER-associated degradation (12). HhN undergoes lipid modifications, in which a cholesterol molecule is covalently linked to the C terminus and a palmitoyl group is linked to the N terminus (13–15). Lipid-modified HhN subsequently exits the ER and is delivered via the secretory pathway to the plasma membrane. Once at the plasma membrane, Hh is released into the extracellular matrix and ultimately recognized by its receptors on the plasma membrane of target cells to induce downstream signal transduction.Although significant progress has been achieved in understanding the Hh signaling pathway in target cells, the molecular mechanisms that mediate secretion of newly synthesized Shh proteins from the producing cells are still unclear. The ER is the first station where newly synthesized proteins enter the secretory pathway. In this compartment, cargo proteins are generally recognized by the coat protein complex II (COPII) to be packaged into vesicles and exported from the ER. Soluble cargo proteins in the ER lumen cannot directly engage the COPII coat but instead are captured into vesicles by transmembrane cargo receptors. One mammalian cargo receptor, ERGIC53, is a mannose-specific lectin that recognizes N-linked glycoproteins in the ER lumen (16, 17). The p24 family of proteins function as cargo receptors to regulate ER export of glycosylphosphatidylinositol (GPI)-anchored proteins (18). Mammalian orthologs of yeast ER vesicle (Erv) proteins have also been thought to function as cargo receptors (16). Surfeit locus protein 4 (SURF4), the mammalian ortholog of Erv29p, regulates ER export of soluble proteins, including lipoproteins and proprotein convertase subtilisin/kexin type 9 (PCSK9) (19–21). SURF4 recognizes amino-terminal tripeptide motifs of soluble cargo proteins and participates in ER exit site (ERES) organization (19, 22). The cargo receptors that mediate sorting of Shh in the secretory pathway remain unknown.Here, we examined trafficking of the N-terminal fragment of Shh without the cholesterol modification (referred to as ShhN). We utilized the Retention Using Selective Hook (RUSH) assay (23) to analyze the kinetics of trafficking of ShhN along the secretory pathway. We reconstituted the packaging of ShhN into transport vesicles in vitro and utilized this assay to quantitatively measure packaging efficiency. Our study reveals cellular factors and underlying mechanisms that mediate the sorting and secretion of Shh, providing insight into the biosynthetic trafficking of Shh.     

Overlapping functions of stonin 2 and SV2 in sorting of the calcium sensor synaptotagmin 1 to synaptic vesicles

Neurotransmission involves the calcium-regulated exocytic fusion of synaptic vesicles (SVs) and the subsequent retrieval of SV membranes followed by reformation of properly sized and shaped SVs. An unresolved question is whether each SV protein is sorted by its own dedicated adaptor or whether sorting is facilitated by association between different SV proteins. We demonstrate that endocytic sorting of the calcium sensor synaptotagmin 1 (Syt1) is mediated by the overlapping activities of the Syt1-associated SV glycoprotein SV2A/B and the endocytic Syt1-adaptor stonin 2 (Stn2). Deletion or knockdown of either SV2A/B or Stn2 results in partial Syt1 loss and missorting of Syt1 to the neuronal surface, whereas deletion of both SV2A/B and Stn2 dramatically exacerbates this phenotype. Selective missorting and degradation of Syt1 in the absence of SV2A/B and Stn2 impairs the efficacy of neurotransmission at hippocampal synapses. These results indicate that endocytic sorting of Syt1 to SVs is mediated by the overlapping activities of SV2A/B and Stn2 and favor a model according to which SV protein sorting is guarded by both cargo-specific mechanisms as well as association between SV proteins.Neurotransmission is based on the calcium-triggered fusion of neurotransmitter-filled synaptic vesicles (SVs) with the presynaptic plasma membrane. To sustain neurotransmitter release, neurons have evolved mechanisms to retrieve SV membranes and to reform SVs locally within presynaptic nerve terminals. How SVs are reformed and maintain their compositional identity (1, 2) is controversial (3–5). One possibility is that upon fusion SV proteins remain clustered at the active zone—that is, by association between SV proteins—and are retrieved via “kiss-and-run” or ultrafast endocytosis (6), thereby alleviating the need for specific sorting of individual SV proteins. Alternatively, if SVs lose their identity during multiple rounds of exo-/endocytosis (7, 8), specific mechanisms exist to orchestrate high-fidelity SV protein sorting, either directly at the plasma membrane via slow clathrin-mediated endocytosis (CME) or at endosome-like vacuoles generated by fast clathrin-independent membrane retrieval (5, 9). Endocytic adaptors for SV protein sorting include the heterotetrameric adaptor protein complex 2 (AP-2) (9), the synaptobrevin 2/VAMP2 adaptor AP180 (10), and the AP-2μ–related protein stonin 2 (Stn2), a specific sorting adaptor for the SV calcium sensor synaptotagmin 1 (Syt1) (8, 11). Although genetic inactivation of the Stn2 orthologs Stoned B and Unc41 in flies and worms is lethal due to defective neurotransmission caused by degradation and missorting of Syt1 (12, 13), Stn2 knockout (KO) mice are viable and able to internalize Syt1, albeit with reduced fidelity of sorting (14). Thus, mammalian synapses, in contrast to invertebrates, have evolved mechanisms to sort Syt1 in the absence of its specific sorting adaptor Stn2. One possibility is that Syt1 sorting in addition to its direct recognition by Stn2 is facilitated by complex formation with other SV proteins. Likely candidates for such a piggyback mechanism are the SV2 family of transmembrane SV glycoproteins (15, 16), which might regulate Syt1 function either via direct interaction (17, 18) or by facilitating its binding to AP-2 (19). Apart from the distantly related SVOP protein (20), no close SV2 homologs exist in invertebrates, suggesting that SV2 fulfills a unique function at mammalian synapses. KO of SV2A or combined loss of its major A and B isoforms in mice causes early postnatal lethality due to epileptic seizures (21, 22), impaired neurotransmission (23, 24), and defects in Syt1 trafficking (25), whereas SV2B KO mice are phenotypically normal (17). Given that SV2A in addition to its association with Syt1 binds to endocytic proteins including AP-2 and Eps15 (25), SV2 would be a likely candidate for mediating Syt1 sorting to SVs.Here we demonstrate that endocytic sorting of Syt1 is mediated by the overlapping activities of SV2A/B and Stn2. Deletion or knockdown of either SV2A/B or Stn2 results in partial Syt1 loss and missorting of Syt1 to the neuronal surface, whereas deletion of both SV2A/B and Stn2 dramatically exacerbates this phenotype, resulting in severely impaired basal neurotransmission. Our results favor a model according to which SV protein sorting is guarded by both cargo-specific mechanisms as well as association between SV proteins.     

An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking

The fidelity of protein transport in the secretory pathway relies on the accurate sorting of proteins to their correct destinations. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on specific sorting machinery to be enriched into transport vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry (MS) analyses of the isolated vesicles revealed cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner. We found that two of them, FAM84B (also known as LRAT domain containing 2 or LRATD2) and PRRC1, contain proline-rich domains and regulate anterograde trafficking. Further analyses revealed that PRRC1 is recruited to endoplasmic reticulum (ER) exit sites, interacts with the inner COPII coat, and its absence increases membrane association of COPII. In addition, we uncovered cargo proteins that depend on GTP hydrolysis to be captured into vesicles. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Our results indicate that the vesicle formation assay in combination with quantitative MS analysis is a robust and powerful tool to uncover novel factors that mediate vesicular trafficking and to uncover cargo clients of specific cellular factors.

The eukaryotic secretory pathway plays important roles in delivering a variety of newly synthesized proteins to their specific resident compartments. The fidelity of protein transport in the secretory pathway depends on accurate sorting of specific cargo proteins into transport vesicles. Defects in cargo sorting cause protein mistargeting and induce defects in establishing cell polarity, immunity, as well as other physiological processes (1).A variety of cytosolic proteins are recruited to the membrane and play important roles in the protein sorting process. These cytosolic proteins include small GTPases of the Arf family and cargo adaptors (1, 2). The Arf family GTPases cycle between a GDP-bound cytosolic state and a GTP-bound state. Upon GTP binding, Arf proteins undergo conformational changes in which the N-terminal amphipathic helix is exposed to bind membranes and the switch domains change their conformation to recruit various cytosolic cargo adaptors. Once recruited onto the membranes, these cargo adaptors recognize sorting motifs on the cargo proteins. This recognition step is important for efficiently capturing cargo proteins into vesicles.The Arf family protein, Sar1, regulates packaging of cargo proteins into vesicles at the endoplasmic reticulum (ER). GTP-bound Sar1 mediates membrane recruitment of the coat protein complex II (COPII) to capture cargo proteins (2). Soluble cargo proteins in the lumen of the ER cannot be directly recognized by COPII coat and such proteins are thought to be linked to the cargo sorting machinery on the cytosolic side by transmembrane cargo receptors. One cargo receptor in mammalian cells, ERGIC53, is a mannose lectin and functions in capturing specific N-linked glycoproteins in the lumen of the ER (3). ERGIC53 regulates ER export of blood coagulation factors V and VIII, a cathepsin-Z–related protein, and alpha1-antittrypsin (4–7). Another cargo receptor, SURF4, binds amino-terminal tripeptide motifs of soluble cargo proteins and regulates ER export of soluble cargo proteins, including the yolk protein VIT-2 in Caenorhabditis elegans (8), and PCSK9 and apolipoprotein B in mammalian cells (9–11).Although significant progress has been made in understanding the general steps of cargo sorting, the spectrum of cargo clients of a specific Arf family member, cargo adaptor, or cargo receptor remains largely underinvestigated. To deepen our understanding of protein sorting in the secretory pathway, it is important to develop a robust approach to systematically reveal cargo proteins that depend on a specific factor to be efficiently packaged into vesicles. Revealing this will provide significant insight into the functions and the specificity of cargo sorting. Since distinct cytosolic proteins are recruited to membranes by different GTP-bound Arf family proteins, systematic approaches are needed to characterize budding events associated with a specific GTP-bound Arf family protein.A cellular imaging approach, pairing analysis of cargo receptors (PAIRS), has been utilized to identify the spectrum of cargo proteins that depend on a specific cargo receptor for ER export in yeast. This analysis focused on around 150 cargo molecules labeled with fluorescent tags (12). An in vitro assay that reconstitutes packaging of cargo proteins into vesicles has been used to reveal protein profiles of vesicles budded with purified COPII or COPI proteins (13). However, this analysis did not identify any non-ER resident transmembrane proteins or secretory proteins (13). This is possibly due to an unappreciated requirement for other cytosolic factors in addition to the COP coats. Affinity chromatography has been utilized to reveal cytosolic proteins that specifically interact with GTP-bound Arf or Rab proteins (14–16). In this approach, the membranes are disrupted, which might preclude identification of membrane-associated effectors. Thus, it is important to develop additional approaches to reveal novel cytosolic proteins that associate with GTP-bound Arf proteins on membranes.Here, we used an in vitro assay to reconstitute packaging of cargo proteins into transport vesicles utilizing rat liver cytosol (RLC) as a source of cytosolic proteins. Analysis of vesicle fractions by quantitative mass spectrometry (MS) revealed cytosolic proteins that are associated with vesicles dependent on GTP or GTP-bound Sar1A, and that regulate protein trafficking. One of the identified proteins, PRRC1, regulates membrane association of the COPII coat and facilitates ER-to-Golgi trafficking. We also revealed cargo proteins that depend on specific cargo receptors, ERGIC53 or SURF4, to be efficiently packaged into vesicles. Our study indicates that the vesicle formation assay is a robust tool to reveal functional roles of specific factors in protein sorting, and to uncover novel factors that regulate vesicular trafficking in the secretory pathway.     

Fog and rain in the Amazon

The diurnal and seasonal water cycles in the Amazon remain poorly simulated in general circulation models, exhibiting peak evapotranspiration in the wrong season and rain too early in the day. We show that those biases are not present in cloud-resolving simulations with parameterized large-scale circulation. The difference is attributed to the representation of the morning fog layer, and to more accurate characterization of convection and its coupling with large-scale circulation. The morning fog layer, present in the wet season but absent in the dry season, dramatically increases cloud albedo, which reduces evapotranspiration through its modulation of the surface energy budget. These results highlight the importance of the coupling between the energy and hydrological cycles and the key role of cloud albedo feedback for climates over tropical continents.Tropical forests, and the Amazon in particular, are the biggest terrestrial CO₂ sinks on the planet, accounting for about 30% of the total net primary productivity in terrestrial ecosystems. Hence, the climate of the Amazon is of particular importance for the fate of global CO₂ concentration in the atmosphere (1). Besides the difficulty of estimating carbon pools (1–3), our incapacity to correctly predict CO₂ fluxes in the continental tropics largely results from inaccurate simulation of the tropical climate (1, 2, 4, 5). More frequent and more intense droughts in particular are expected to affect the future health of the Amazon and its capacity to act as a major carbon sink (6–8). The land surface is not isolated, however, but interacts with the weather and climate through a series of land−atmosphere feedback loops, which couple the energy, carbon, and water cycles through stomata regulation and boundary layer mediation (9).Current General Circulation Models (GCMs) fail to correctly represent some of the key features of the Amazon climate. In particular, they (i) underestimate the precipitation in the region (10, 11), (ii) do not reproduce the seasonality of either precipitation (10, 11) or surface fluxes such as evapotranspiration (12), and (iii) produce errors in the diurnal cycle and intensity of precipitation, with a tendency to rain too little and too early in the day (13, 14). In the more humid Western part of the basin, surface incoming radiation, evapotranspiration, and photosynthesis all tend to peak in the dry season (15–17), whereas GCMs simulate peaks of those fluxes in the wet season (10, 11). Those issues might be related to the representation of convection (1, 2, 4, 5, 13, 14) and vegetation water stress (6–8, 15–17) in GCMs.We here show that we can represent the Amazonian climate using a strategy opposite to GCMs in which we resolve convection and parameterize the large-scale circulation (Methods). The simulations lack many of the biases observed in GCMs and more accurately capture the differences between the dry and wet season of the Amazon in surface heat fluxes and precipitation. Besides top-of-the-atmosphere insolation, the simulations require the monthly mean temperature profile as an input. We demonstrate that this profile, whose seasonal cycle itself is a product of the coupled ocean−land−atmosphere dynamics, mediates the seasonality of the Amazonian climate by modulating the vertical structure of the large-scale circulation in such a way that thermal energy is less effectively ventilated in the rainy season.     

Membrane insertion mechanism of the caveola coat protein Cavin1

Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades, and lipid sorting. The caveola coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modeling to show that Cavin1 inserts into membranes. We establish that initial phosphatidylinositol (4, 5) bisphosphate [PI(4,5)P₂]–dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the coassembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane.

The typical small bulb-shaped invaginations of the plasma membrane termed “caveolae” are found in most vertebrate cells. They are highly abundant in adipocytes, muscle, and endothelial cells and are important for various physiological processes like regulation of membrane tension, lipid metabolism, and cellular signaling (1, 2). Lack or dysfunction of caveolae is connected to severe human diseases such as muscular dystrophy, cardiomyopathy, and lipodystrophy. Caveolae formation is dependent on membrane lipid composition and the coat components Caveolin1 (CAV1) and Cavin1 (3). Caveolae are enriched in cholesterol and sphingolipids (1, 2), which not only accumulate in caveolae but are actively sequestered (4). The negatively charged lipids phosphatidylserine (PS) and phosphatidylinositol (4, 5) bisphosphate [PI(4,5)P₂] are also enriched in caveolae (5). Lipid mapping in cells showed that both CAV1 and Cavin1 recruit specific lipid species to caveolae, hereby acting synergistically to generate the unique lipid nanoenvironment of caveolae (6, 7). CAV1 and Cavin1 are universal structural elements, and knockout of either of these proteins leads to loss of caveolae (1, 2). Electron microscopy studies on caveolae have revealed a striated protein coat lining, which is believed to comprise CAV1 and the cavin proteins (8, 9). CAV1 belongs to a family of integral membrane proteins (CAV1 to 3), where both the N and C termini protrude into the cytoplasm. CAV1 has been shown to form high-order 8S oligomers in membranes following cholesterol binding (10). Cavin1 belongs to a family composed of four different proteins (Cavin1 to 4), which exhibit tissue-specific expression patterns (3). The cavin proteins are thought to assemble with CAV1 8S complexes to form 60S and 80S complexes building up the caveola coat (11). Importantly, Cavin1 is required for membrane invagination of caveolae (12). Cryoelectron microscopy studies of such complexes proposed an architecture composed of an inner cage of polygonal units of caveolins and an outer cavin coat (13, 14). The models propose that cavin arranges into a web-like architecture composed of an interbranched trimeric complex (13) or alternatively that the cavins are stacked in rod-like trimers (14). However, it is still not understood how the unique striped or spiral pattern of the caveola coat is assembled and what intermolecular forces join the molecular components together.The cavin proteins share a common pattern in their domain structure, containing negatively charged disordered regions (DRs) interspersed with positively charged helical regions (HRs) (Fig. 1A). The crystal structures of HR1 (Protein Data Bank [PDB] ID codes 4QKV and 4QKW) revealed an extended α-helical trimeric coiled-coil structure (15). The HR1 domain has been shown to mediate trimeric homooligomerization of Cavin1 and formation of heterocomplexes with either Cavin2 or Cavin3 in solution (15, 16). HR2 is also thought to build up a trimeric coiled coil, but this structural arrangement is dependent on HR1. In vitro studies have shown that Cavin1 binds both PI(4,5)P₂ and PS (15, 17). The positively charged amino acids (Lys115, Arg117, Lys118, Lys124, Arg127) in the HR1 domain mediate specific binding to PI(4,5)P₂ (15), whereas a repeated sequence of 11 amino acids of the HR2 domain, identified as an undecad repeat (UC1), was shown to bind PS (17). Furthermore, Cavin1 has been shown to generate membrane curvature in vitro (15). Both HRs and DRs were required for this, and it was proposed that Cavin1 drives membrane curvature by molecular crowding via weak electrostatic interactions between the DRs and HRs (18). Interestingly, the assembly of both CAV1 and Cavin1 was found to be dependent on the acyl chain composition of PS, suggesting that Cavin1 might also interact with the hydrophobic region of the membrane (6). Membrane insertion of Cavin1 could contribute to membrane curvature generation and the formation of caveolae. Yet, based on the current structural understanding, it is not clear how Cavin1 orients and assembles at the membrane interface.Open in a separate windowFig. 1.Cavin1 binding and insertion into model lipid membranes. (A) Scheme of the domain structure of Cavin1 with DRs and HRs. White stripes mark undecad repeats. The crystal structure of HR1 (PDB ID code 4QKV) is displayed (Top). Regions involved in binding to PI(4,5)P₂, PS, and CAV1 are indicated. (B) Liposome cosedimentation of Cavin1. Cavin1 was incubated with or without DOPC:DOPE:PI(4,5)P₂ liposomes and centrifuged, and supernatant (S) and pellet (P) fractions were analyzed by SDS-PAGE. Band intensities were quantified and data are shown as mean ± SEM (n = 3). (C, Top) Scheme of SLB formation. (C, Bottom) QCM-D measurement showing a shift in frequency (ΔF) (black line) and dissipation (ΔD) (red line) upon SLB formation. (D, Top) Illustration of QCM-D setup. (D, Bottom) QCM-D monitoring of Cavin1 adsorption to an SLB. The responses in ΔF and ΔD correspond to Cavin1 injection and buffer rinses as indicated. The gray dotted line shows extrapolation of protein desorption from the first rinse (150 mM NaCl). (E, Top) Scheme of monolayer protein adsorption experiments. (E, Bottom) Cavin1 adsorption to DOPC:DOPE:PI(4,5)P₂ monolayers. Cavin1 was injected underneath the film at π₀ = 20 mN⋅m⁻¹ and Δπ was recorded over time. (F) Cavin1 adsorption to lipid monolayers was measured at different π₀. The MIP value was determined by extrapolation of the Δπ/π₀ plot to the x axis.In this work, we address the detailed mechanism by which Cavin1 binds and assembles at the lipid interface using model membranes in combination with a variety of biophysical techniques. We found that Cavin1 inserted into the membrane via the HR1 domain in a PI(4,5)P₂-mediated process. Membrane insertion involved partial separation of the helices in the HR1 domains in a process aided by the DR domains.     

Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes

Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu²⁺ binding site and defined cross-dimer copper–copper distance distributions. Importantly, we find that copper deficiency in these mutants promotes aggregation in a manner strikingly consistent with their clinical severities. G93 mutants seem to properly incorporate metal ions under physiological conditions when assisted by the copper chaperone but release copper under destabilizing conditions more readily than the WT enzyme. Altered intradimer flexibility in ALS mutants may cause differential metal retention and promote distinct aggregation trends observed for mutant proteins in vitro and in ALS patients. Combined biophysical and structural results test and link copper retention to the framework destabilization hypothesis as a unifying general mechanism for both SOD aggregation and ALS disease progression, with implications for disease severity and therapeutic intervention strategies.ALS is a lethal degenerative disease of the human motor system (1). Opportunities for improved understanding and clinical intervention arose from the discovery that up to 23.5% of familial ALS cases and 7% of spontaneous cases are caused by mutations in the superoxide dismutase 1 (SOD1) gene encoding human Cu, Zn SOD (2–4). SOD is a highly conserved (5), dimeric, antioxidant metalloenzyme that detoxifies superoxide radicals (6, 7), but overexpression of SOD1 ALS mutants is sufficient to cause disease in mice (8). Misfolded and/or aggregated SOD species are deposited within mouse neuronal and glial inclusions (9, 10), even before symptoms appear (11, 12). Although human familial ALS has a symptomatic phenotype indistinguishable from sporadic cases (13), individual SOD1 mutations can result in highly variable disease progression and penetrance (14, 15).Many nongeneral mechanisms, including loss of activity or gain of function, were postulated to explain the roles of SOD mutants in ALS (3, 16–19). Recently, however, an initial hypothesis proposing that SOD manifests disease symptoms by framework destabilization (protein instability caused by structural defects) and consequent protein misassembly and aggregation has gained renewed support (2, 10, 14, 20–23). Ironically, WT SOD is an unusually stable protein (7, 24–26), and precisely how SOD mutations cause disease remains unclear. For instance, human SOD free cysteine residues C6 and C111 have been implicated in protein aggregation by promoting cross-linking (27, 28) and/or stability changes associated with oxidative modifications (29–33). Mutation of the chemically reactive thiols significantly decreases the irreversible denaturation rate for human and bovine SOD (24, 34). However, ALS mutants in a C6A/C111S SOD (AS-SOD) background (35, 36) maintain the native C57–C146 disulfide bond but can still undergo aggregation, and mutations of the free cysteines can cause ALS (37, 38). These results imply that free cysteines are not strictly required but rather, may alter aggregation kinetics (20). SOD also contains two metal ion cofactors in each subunit: a catalytic copper ion (6) and a structurally stabilizing zinc ion (34, 39, 40) (Fig. 1A). In higher eukaryotes, a copper chaperone for SOD (CCS) plays an important role in catalyzing both the copper incorporation and native disulfide bond formation (41). Structural analyses of apo WT SOD point to greater flexibility or increased solvent accessibility of C6 otherwise buried in the stable dimer interface (42, 43), and molecular dynamics simulations also suggest a critical role for metal ions in protein structure, because SOD’s β-sheet propensity decreases in the absence of metals (44). As a result, apo SOD readily forms protein aggregates (45, 46), but the molecular structures of SOD aggregates are likely polymorphic and represent a controversial topic (23, 47–51). The intertwined effects of the aggregation-enhancing free cysteines, dimer-stabilizing metal ions, and CCS maturation of SOD complicate the study of the ALS-causing SOD mutations themselves, and therefore, a clear cause-and-effect relationship remains obscure and requires deconvolution.Open in a separate windowFig. 1.Comparison of crystallographic and solution structures of WT and G93A SOD. (A) Overall architecture of the WT SOD dimer is displayed in 90° rotated views. G93 (small red spheres) resides on a surface-exposed interstrand loop between the fifth and sixth sequential β-strands of SOD and is expected to be innocuous in facilitating protein stability; however, this site harbors the most substitutions observed to result in ALS. G93 is also distant from both (Upper) the dimer interface and (Lower Left) the SOD active site (gold and silver spheres), which are generally implicated as the major determinants for SOD stability. Small blue spheres denote free cysteines. (Lower Right) The close-up view of the mutation site (boxed region in Lower Left tilted forward) shows high similarity between WT (purple) and G93A (red) SOD crystal structures [Protein Data Bank ID codes 1PU0 (WT) and 2ZKY (G93A)]. Hydrogen bonds characteristic of a β-bulge motif are indicated, whereby G93 (or A93) represents position 1. The main chain carbonyl group of β-barrel cork residue L38 is adjacent to the G93 site. (B) SAXS-derived electron pair P(r) distributions from WT (purple) and G93A (red) SOD samples in solution are compared with the theoretical curve for 1PU0. P(r) plots are normalized to peak height. Ab initio models of WT SOD derived from P(r) data are depicted in purple, with crystal structure docked into mesh envelope. Contributions to major and minor peaks from subunit and dimer dimensions are indicated.To better understand the structural effects of ALS mutations on SOD architecture, we coupled the wealth of crystallographic knowledge on SOD structure (7, 52, 53) with small-angle X-ray scattering (SAXS) experiments to characterize misassembly aggregates of ALS mutant SODs in solution. Over 20 y ago, we solved the first atomic structure of the human WT SOD protein (Fig. 1A) (20, 34) and proposed the framework destabilization hypothesis to explain how diverse mutations located throughout the 153-residue β-barrel enzyme might produce a similar disease phenotype (2), albeit with distinctions in the progression trajectory. Since that time, a staggering number of ALS mutations has been documented in patients [178 (mostly missense) (54)], with a similar phenotype in dogs (55, 56). Solution-based techniques are increasingly being applied to connect structure to biological outcome, for instance, through examination of intermolecular interactions within stress-activated pathways, for instance (57, 58). SAXS, which can probe structures for a wide size range of species, also provides higher resolution insights (59), for instance, over visible light-scattering techniques, readily distinguishing unfolded from folded proteins (60).Here, we monitor the initial events of protein aggregation in a subset of ALS mutants localized to a mutational hotspot site at glycine 93. Specifically, we wished to test a possible structural basis for how G93 mutations (to A, C, D, R, S, or V) modulate age of onset and clinical severity in ALS patients (14, 15). The G93 substitution occurs in a β-bulge region (61) between sequential β-strands of the protein (Fig. 1A) on a protruding loop roughly ∼20 Å from T54, the nearest residue of the opposing subunit, and the metal-containing active site (Fig. S1). A priori, mutation of this outer loop position would not be expected to interfere with active site chemistry or buried molecular interfaces. However, we discovered correlations of aggregation nucleation kinetics of SOD proteins with ALS mutations at this site, the stabilizing effects of metal ion retention, and available data for clinical phenotypes in patients with the same mutation. Furthermore, by measuring and exploiting the dimer geometry to observe intrinsic SOD conformers, we show that G93 mutant proteins natively reveal increased intradimer conformational flexibility in the absence of aggregation, which may reflect an increased tendency for ALS mutants to become metal-deficient and misfolding-prone and further explain the correlation to disease severity. Collective results on G93 mutants, thus, support and extend the framework destabilization hypothesis.     

Insect’s intestinal organ for symbiont sorting

Symbiosis has significantly contributed to organismal adaptation and diversification. For establishment and maintenance of such host–symbiont associations, host organisms must have evolved mechanisms for selective incorporation, accommodation, and maintenance of their specific microbial partners. Here we report the discovery of a previously unrecognized type of animal organ for symbiont sorting. In the bean bug Riptortus pedestris, the posterior midgut is morphologically differentiated for harboring specific symbiotic bacteria of a beneficial nature. The sorting organ lies in the middle of the intestine as a constricted region, which partitions the midgut into an anterior nonsymbiotic region and a posterior symbiotic region. Oral administration of GFP-labeled Burkholderia symbionts to nymphal stinkbugs showed that the symbionts pass through the constricted region and colonize the posterior midgut. However, administration of food colorings revealed that food fluid enters neither the constricted region nor the posterior midgut, indicating selective symbiont passage at the constricted region and functional isolation of the posterior midgut for symbiosis. Coadministration of the GFP-labeled symbiont and red fluorescent protein-labeled Escherichia coli unveiled selective passage of the symbiont and blockage of E. coli at the constricted region, demonstrating the organ’s ability to discriminate the specific bacterial symbiont from nonsymbiotic bacteria. Transposon mutagenesis and screening revealed that symbiont mutants in flagella-related genes fail to pass through the constricted region, highlighting that both host’s control and symbiont’s motility are involved in the sorting process. The blocking of food flow at the constricted region is conserved among diverse stinkbug groups, suggesting the evolutionary origin of the intestinal organ in their common ancestor.Diverse organisms are obligatorily associated with microbial symbionts, which significantly contribute to their adaptation and survival (1–3). In such symbiotic associations, the host organisms often develop specialized cells, tissues, or organs for harboring their specific microbial partners [for example, root nodules in the legume–Rhizobium symbiosis (4, 5), symbiotic light organs in the squid–Vibrio symbiosis (6, 7), and bacteriocytes in the aphid–Buchnera symbiosis (8, 9)].These microbial symbionts are either acquired by newborn hosts from the environment every generation as in the legume–Rhizobium and the squid–Vibrio symbioses or transmitted vertically through host generations as in the aphid–Buchnera symbiosis (10). In the environmentally acquired symbiotic associations, it is essential for the host organisms to recognize and incorporate specific symbiotic bacteria while excluding a myriad of nonsymbiotic environmental microbes (6, 11). In the vertically transmitted symbiotic associations, it is important for the host organisms to selectively transmit their own symbiotic bacteria while excluding parasitic/cheating microbial contaminants (12–14). Hence, it is expected that the host organisms must have evolved some mechanisms for selective incorporation, accommodation, and maintenance of their specific microbial partners. Those controlling mechanisms are of general importance for understanding symbiosis (6, 10).Stinkbugs, belonging to the insect order Hemiptera, consist of over 40,000 described species in the world (15). The majority of the stinkbugs suck plant sap or tissues, and some of them are notorious as devastating agricultural pests (16). These plant-sucking stinkbugs possess a specialized symbiotic organ in their alimentary tract: A posterior region of the midgut is morphologically differentiated with a number of sacs or tubular outgrowths, called crypts or ceca, whose inner cavity hosts symbiotic bacteria (17–21). Usually, a single bacterial species dominates in the midgut crypts, and elimination of the symbiont causes retarded growth and increased mortality of the host, which indicates the specific and beneficial nature of the stinkbug gut symbiosis (20–31). The initial symbiont infection is established by nymphal feeding, which may be either via vertical transmission from symbiont-containing maternal secretion supplied upon oviposition (19–21) or via environmental acquisition from ambient microbiota (21–23). What mechanisms underlie the selective establishment of a specific bacterial symbiont in the midgut symbiotic organ despite the oral inoculum contaminated by nonsymbiotic microbes has remained largely an enigma, although recent studies have started to shed light on the symbiotic mechanisms underlying the environmental acquisition of specific Burkholderia symbionts in the bean bug Riptortus pedestris (Hemiptera: Alydidae) (22, 32). Antimicrobial substances produced by the midgut epithelia (33, 34) and some symbiont factors, such as stress-responsive polyester accumulation, cell wall synthesis, and purine biosynthesis (35–37) might be involved in the selective infection of the Burkholderia symbiont to the midgut crypts.Here we address this important symbiotic issue by the discovery of a previously unrecognized intestinal organ in the stinkbugs. Though very tiny and inconspicuous, the organ governs the configuration and specificity of the stinkbug gut symbiosis. Lying in the middle of the midgut, the organ blocks food flow and nonsymbiotic bacteria but selectively allows passing of specific symbiotic bacteria, whereby the stinkbug’s intestine is functionally partitioned into the anterior region specialized for digestion and absorption and the posterior region dedicated to symbiosis. The blocking of food flow by the organ is conserved across diverse stinkbug families, suggesting the possibility that the organ evolved in their common ancestor and has played substantial roles in their symbiont-mediated adaptation and diversification.     

Two-dimensional electronic–vibrational sum frequency spectroscopy for interactions of electronic and nuclear motions at interfaces

Interactions of electronic and vibrational degrees of freedom are essential for understanding excited-states relaxation pathways of molecular systems at interfaces and surfaces. Here, we present the development of interface-specific two-dimensional electronic–vibrational sum frequency generation (2D-EVSFG) spectroscopy for electronic–vibrational couplings for excited states at interfaces and surfaces. We demonstrate this 2D-EVSFG technique by investigating photoexcited interface-active (E)-4-((4-(dihexylamino) phenyl)diazinyl)-1-methylpyridin-1- lum (AP3) molecules at the air–water interface as an example. Our 2D-EVSFG experiments show strong vibronic couplings of interfacial AP3 molecules upon photoexcitation and subsequent relaxation of a locally excited (LE) state. Time-dependent 2D-EVSFG experiments indicate that the relaxation of the LE state, S₂, is strongly coupled with two high-frequency modes of 1,529.1 and 1,568.1 cm⁻¹. Quantum chemistry calculations further verify that the strong vibronic couplings of the two vibrations promote the transition from the S₂ state to the lower excited state S₁. We believe that this development of 2D-EVSFG opens up an avenue of understanding excited-state dynamics related to interfaces and surfaces.

Electronic and vibrational degrees of freedom are the most important physical quantities in molecular systems at interfaces and surfaces. Knowledge of interactions between electronic and vibrational motions, namely electronic–vibrational couplings, is essential to understanding excited-states relaxation pathways of molecular systems at interfaces and surfaces. Many excited-states relaxation processes occur at interfaces and surfaces, including charge transfer, energy transfer, proton transfer, proton-coupled electron transfer, configurational dynamics, and so on (1–11). These relaxation processes are intimately related to the electronic–vibrational couplings at interfaces and surfaces. Strong electronic–vibrational couplings could promote nonadiabatic evolution of excited potential energy and thus, facilitate chemical reactions or intramolecular structural changes of interfacial molecules (10, 12, 13). Furthermore, these interactions of electronic and vibrational degrees of freedom are subject to solvent environments (e.g., interfaces/surfaces with a restricted environment of unique physical and chemical properties) (9, 14, 15). Despite the importance of interactions of electronic and vibrational motions, little is known about excited-state electronic–vibrational couplings at interfaces and surfaces.Interface-specific electronic and vibrational spectroscopies enable us to characterize the electronic and vibrational structures separately. As interface-specific tools, second-order electronic sum frequency generation (ESFG) and vibrational sum frequency generation (VSFG) spectroscopies have been utilized for investigating molecular structure, orientational configurations, chemical reactions, chirality, static potential, environmental issues, and biological systems at interfaces and surfaces (16–52). Recently, structural dynamics at interfaces and surfaces have been explored using time-resolved ESFG and time-resolved VSFG with a visible pump or an infrared (IR) pump thanks to the development of ultrafast lasers (6–9, 13–15, 49, 53–61). Doubly resonant sum frequency generation (SFG) has been demonstrated to probe both electronic and vibration transitions of interfacial molecular monolayer (15, 62–71). This frequency-domain two-dimensional (2D) interface/surface spectroscopy could provide information regarding electronic–vibrational coupling of interfacial molecules. However, contributions from excited states are too weak to be probed due to large damping rates of vibrational states in excited states (62, 63). As such, the frequency-domain doubly resonant SFG is used only for electronic–vibrational coupling of electronic ground states. Ultrafast interface-specific electronic–vibrational spectroscopy could allow us to gain insights into how specific nuclear motions drive the relaxation of electronic excited states. Therefore, development of interface-specific electronic–vibrational spectroscopy for excited states is needed.In this work, we integrate the specificity of interfaces and surfaces into the capabilities of ultrafast 2D spectroscopy for dynamical electronic–vibrational couplings in excited states of molecules; 2D interface-specific spectroscopies are analogous to those 2D spectra in bulk that spread the information contained in a pump−probe spectrum over two frequency axes. Thus, one can better interpret congested one-dimensional signals. Two-dimensional vibrational sum frequency generation (2D-VSFG) spectroscopy was demonstrated a few year ago (72–74). Furthermore, heterodyne 2D-VSFG spectroscopy using middle infrared (mid-IR) pulse shaping and noncollinear geometry 2D-VSFG experiments have also been developed to study vibrational structures and dynamics at interfaces (31, 75–78). Recently, two-dimensional electronic sum frequency generation (2D-ESFG) spectroscopy has also been demonstrated for surfaces and interfaces (79). On the other hand, bulk two-dimensional electronic–vibrational (2D-EV) spectroscopy has been extensively used to investigate the electronic relaxation and energy transfer dynamics of molecules, biological systems, and nanomaterials (80–90). The 2D-EV technique not only provides electronic and vibrational interactions between excitons or different excited electronic states of systems but also, identifies fast nonradiative transitions through nuclear motions in molecules, aggregations, and nanomaterials. However, an interface-specific technique for two-dimensional electronic–vibrational sum frequency generation (2D-EVSFG) spectroscopy has yet to be developed.Here, we present the development of 2D-EVSFG spectroscopy for the couplings of electronic and nucleic motions at interfaces and surfaces. The purpose of developing 2D-EVSFG spectroscopy is to bridge the gap between the visible and IR regions to reveal how structural dynamics for photoexcited electronic states are coupled with vibrations at interfaces and surfaces. As an example, we applied this 2D-EVSFG experimental method to time evolution of electronic–vibrational couplings at excited states of interface-active molecules at the air–water interface.     

A mechanism for retromer endosomal coat complex assembly with cargo

Retromer is an evolutionarily conserved protein complex composed of the VPS26, VPS29, and VPS35 proteins that selects and packages cargo proteins into transport carriers that export cargo from the endosome. The mechanisms by which retromer is recruited to the endosome and captures cargo are unknown. We show that membrane recruitment of retromer is mediated by bivalent recognition of an effector of PI3K, SNX3, and the RAB7A GTPase, by the VPS35 retromer subunit. These bivalent interactions prime retromer to capture integral membrane cargo, which enhances membrane association of retromer and initiates cargo sorting. The role of RAB7A is severely impaired by a mutation, K157N, that causes Charcot–Marie–Tooth neuropathy 2B. The results elucidate minimal requirements for retromer assembly on the endosome membrane and reveal how PI3K and RAB signaling are coupled to initiate retromer-mediated cargo export.Sorting of cargo within the endosome determines whether it will be retained and ultimately degraded via lysosome-mediated turnover, or exported via a plasma membrane recycling or retrograde pathway that directs cargo to the TGN or recycling endosome. Genetic dissection of endosomal retrograde pathways in budding yeast (Saccharomyces cerevisiae) led to the identification of an endosome-associated protein complex termed retromer, composed of a Vps5–Vps17 heterodimer and a trimeric complex of the Vps26, Vps29, and Vps35 proteins (1). The retromer trimer, also called the “cargo recognition complex,” is the core functional unit of retromer, serving as a platform for recruiting many other factors to the endosome (2), and we shall refer herein to the trimer as retromer. It is now appreciated that retromer constitutes an ancient, evolutionarily conserved protein sorting complex that operates in multiple endosomal cargo export pathways (2, 3). Hence, elucidating the molecular mechanisms that underlie retromer function is key for understanding the endosomal system.The formation of a vesicular transport carrier is typically initiated by a GTPase module that elicits recruitment of a coat protein from the cytosol to a particular site on the membrane. Retromer is an effector of RAB7A [henceforth referred to as RAB7 (human) or Ypt7 (yeast)], a GTPase regulator of endosome dynamics and depletion of RAB7-GTP in cells results in a substantial loss of endosome-associated retromer (4–9). In addition to GTPase signaling modules, interactions of coat proteins with membrane lipids, such as phosphoinositides, contribute to coat assembly by increasing the avidity of membrane binding. There is no evidence that retromer directly recognizes membrane lipids (10, 11). Instead, retromer membrane recruitment is attributed to its association with any of several different sorting nexins (3), which are peripheral membrane proteins defined by the presence of a Phox homology (PX) domain that recognizes phosphatidylinositol 3-phosphate (PtdIns3P), a signature component of endosomal membranes. However, a formal test of this hypothesis is lacking. Retromer binds sorting nexin 3 [henceforth referred to as SNX3 (human) or Snx3 (yeast)] (12–15), and in SNX3 knockdown cells, less retromer is associated with endosomes (14). In this study, we show that SNX3 and RAB7 are coordinately recognized by retromer and that these interactions are sufficient to recruit retromer to a membrane where they poise retromer to capture integral membrane retrograde cargo.     

Thermodynamics of formation of coffinite,USiO4

Coffinite, USiO₄, is an important U(IV) mineral, but its thermodynamic properties are not well-constrained. In this work, two different coffinite samples were synthesized under hydrothermal conditions and purified from a mixture of products. The enthalpy of formation was obtained by high-temperature oxide melt solution calorimetry. Coffinite is energetically metastable with respect to a mixture of UO₂ (uraninite) and SiO₂ (quartz) by 25.6 ± 3.9 kJ/mol. Its standard enthalpy of formation from the elements at 25 °C is −1,970.0 ± 4.2 kJ/mol. Decomposition of the two samples was characterized by X-ray diffraction and by thermogravimetry and differential scanning calorimetry coupled with mass spectrometric analysis of evolved gases. Coffinite slowly decomposes to U₃O₈ and SiO₂ starting around 450 °C in air and thus has poor thermal stability in the ambient environment. The energetic metastability explains why coffinite cannot be synthesized directly from uraninite and quartz but can be made by low-temperature precipitation in aqueous and hydrothermal environments. These thermochemical constraints are in accord with observations of the occurrence of coffinite in nature and are relevant to spent nuclear fuel corrosion.In many countries with nuclear energy programs, spent nuclear fuel (SNF) and/or vitrified high-level radioactive waste will be disposed in an underground geological repository. Demonstrating the long-term (10⁶–10⁹ y) safety of such a repository system is a major challenge. The potential release of radionuclides into the environment strongly depends on the availability of water and the subsequent corrosion of the waste form as well as the formation of secondary phases, which control the radionuclide solubility. Coffinite (1), USiO₄, is expected to be an important alteration product of SNF in contact with silica-enriched groundwater under reducing conditions (2–8). It is also found, accompanied by thorium orthosilicate and uranothorite, in igneous and metamorphic rocks and ore minerals from uranium and thorium sedimentary deposits (2, 4, 5, 8–16). Under reducing conditions in the repository system, the uranium solubility (very low) in aqueous solutions is typically derived from the solubility product of UO₂. Stable U(IV) minerals, which could form as secondary phases, would impart lower uranium solubility to such systems. Thus, knowledge of coffinite thermodynamics is needed to constrain the solubility of U(IV) in natural environments and would be useful in repository assessment.In natural uranium deposits such as Oklo (Gabon) (4, 7, 11, 12, 14, 17, 18) and Cigar Lake (Canada) (5, 13, 15), coffinite has been suggested to coexist with uraninite, based on electron probe microanalysis (EPMA) (4, 5, 7, 11, 13, 17, 19, 20) and transmission electron microscopy (TEM) (8, 15). However, it is not clear whether such apparent replacement of uraninite by a coffinite-like phase is a direct solid-state process or occurs mediated by dissolution and reprecipitation.The precipitation of USiO₄ as a secondary phase should be favored in contact with silica-rich groundwater (21) [silica concentration >10⁻⁴ mol/L (22, 23)]. Natural coffinite samples are often fine-grained (4, 5, 8, 11, 13, 15, 24), due to the long exposure to alpha-decay event irradiation (4, 6, 25, 26) and are associated with other minerals and organic matter (6, 8, 12, 18, 27, 28). Hence the determination of accurate thermodynamic data from natural samples is not straightforward. However, the synthesis of pure coffinite also has challenges. It appears not to form by reacting the oxides under dry high-temperature conditions (24, 29). Synthesis from aqueous solutions usually produces UO₂ and amorphous SiO₂ impurities, with coffinite sometimes being only a minor phase (24, 30–35). It is not clear whether these difficulties arise from kinetic factors (slow reaction rates) or reflect intrinsic thermodynamic instability (33). Thus, there are only a few reported estimates of thermodynamic properties of coffinite (22, 36–40) and some of them are inconsistent. To resolve these uncertainties, we directly investigated the energetics of synthetic coffinite by high-temperature oxide melt solution calorimetry to obtain a reliable enthalpy of formation and explored its thermal decomposition.     

Cryo-EM structure of the needle filament tip complex of the Salmonella type III secretion injectisome

Type III secretion systems are multiprotein molecular machines required for the virulence of several important bacterial pathogens. The central element of these machines is the injectisome, a ∼5-Md multiprotein structure that mediates the delivery of bacterially encoded proteins into eukaryotic target cells. The injectisome is composed of a cytoplasmic sorting platform, and a membrane-embedded needle complex, which is made up of a multiring base and a needle-like filament that extends several nanometers from the bacterial surface. The needle filament is capped at its distal end by another substructure known as the tip complex, which is crucial for the translocation of effector proteins through the eukaryotic cell plasma membrane. Here we report the cryo-EM structure of the Salmonella Typhimurium needle tip complex docked onto the needle filament tip. Combined with a detailed analysis of structurally guided mutants, this study provides major insight into the assembly and function of this essential component of the type III secretion protein injection machine.

Many pathogenic or symbiotic bacteria for plants or animals have evolved specialized molecular machines known as type III protein secretion systems (T3SSs) (1–3). These machines inject bacterially encoded effector proteins into target eukaryotic cells to modulate cellular processes and ensure the survival and replication of the pathogens or symbionts that encode them (4–6). Although the structural organization of this secretion machine has been largely derived from studies of the T3SSs of the bacterial pathogens Salmonella Typhimurium and Shigella flexneri (7–15), given the conservation of its core components, it is predicted that the T3SSs in other bacteria exhibit a similar architecture. The secretion machine itself is composed of a ∼5-Md multiprotein assembly known as the injectisome (1, 7, 14, 16, 17). This core structure consists of two large substructures, an envelope-associated needle complex (7, 16, 18, 19) and a large cytoplasmic complex known as the “sorting platform” (14, 15, 20).The needle complex is composed of a multiring hollow base, which anchors the injectisome to the bacterial envelope (18). The base encloses the export apparatus, a helical structure that serves as a conduit for the passage through the inner membrane of the proteins destined to transit this secretion pathway (21). The base is also linked to a needle-like filament, which protrudes several nanometers from the bacterial surface and is formed by a single protein arranged in a helical fashion (17, 22). The needle filament, which is traversed in its entire length by a central channel ∼3 nm in diameter, is linked to the base through a structure known as the inner rod (11) and is capped at its distal end by another substructure known as the “tip complex” (23, 24).The sorting platform is located in its entirety within the cytoplasm (14, 15, 20). It is made of six pods that form a cage-like enclosure, which is capped on its cytoplasmic side by a wheel-like structure that holds a hexameric ATPase. Also harbored within the sorting platform cage is the large cytoplasmic domain of one of the components of the export apparatus, which is arranged as two concentric rings and forms a conduit for the secreted substrates to reach the entrance of the export channel (25).A distinctive feature of the T3SSs is that their activation requires contact with the target eukaryotic cell (26, 27). The activation of the T3SS is followed by the deployment of the translocon substructure, which firmly anchors the injectisome to the target cell and serves as the passageway for the effector proteins across the eukaryotic cell membrane. Although little is known about the activation process, it is thought that sensing of the target cell by the tip complex initiates a signaling event that is transduced to the secretion machine by the needle filament itself (26–31). Activation of the secretion machine is then followed by the deployment of the translocon on the target cell membrane, which along with the tip complex and the needle filament, form a continuous passageway through which effector proteins transit from the bacterial cytoplasm to the cytosol of the eukaryotic cell (23, 32, 33). The composition of the tip complex has been the subject of some controversy. While it has been proposed that in the T3SSs of Yersinia spp., Pseudomonas aeruginosa, and Salmonella spp. the tip structure is made up of a single protein, LcrV (24), PcrV (34), and SipD (35), respectively, in the case of Shigella spp., it has been alternatively proposed to be composed of two proteins, IpaB and IpaD (36), or just IpaD (37, 38). The crystal structures of monomeric SipD and close homologs show that these proteins are arranged in three domains: an N-terminal α-helical hairpin, a central coiled-coil, and a mixed α/β carboxyl-terminal domain (39–42). It has been proposed that the N-terminal α-helical hairpin domain functions as a self-chaperone that prevents the self-oligomerization and/or the premature interaction of the tip protein with the needle filament subunit within the bacterial cytoplasm (39). A current hypothesis is that during assembly at the tip of the needle, the N-terminal α-helical hairpin of SipD/IpaD is displaced to allow other domains to interact with the needle. However, there is no structural information of the fully assembled tip complex to support this hypothesis. How the tip protein assembles into the tip complex, and how it is anchored at the distal end of the needle filament, is currently unknown in large part because of the absence of a high-resolution structure of this complex. Understanding of the events that lead to the activation of the secretion machine requires detailed knowledge not only of the structure of the tip complex that caps the needle filament but, importantly, its interface with the needle filament itself.Advances in cryoelectron microscopy (cryo-EM) have allowed the visualization of most components of the T3SS machine at high resolution, both in isolation as well as in situ (7–15). However, the tip structure has eluded high-resolution visualization, in part because existing needle complex isolation protocols result in the dissociation of the tip complex from the needle filament. Here we report the visualization at high resolution by cryo-EM of the tip structure of the needle complex of the S. Typhimurium T3SS encoded within its pathogenicity island 1. Combined with functional analysis, the structure provides major insight into the potential mechanisms of injectisome assembly and activation and fills one of the remaining gaps in the quest for the high-resolution visualization of the entire T3SS injectisome.     

Computational redesign of the lipid-facing surface of the outer membrane protein OmpA

Advances in computational design methods have made possible extensive engineering of soluble proteins, but designed β-barrel membrane proteins await improvements in our understanding of the sequence determinants of folding and stability. A subset of the amino acid residues of membrane proteins interact with the cell membrane, and the design rules that govern this lipid-facing surface are poorly understood. We applied a residue-level depth potential for β-barrel membrane proteins to the complete redesign of the lipid-facing surface of Escherichia coli OmpA. Initial designs failed to fold correctly, but reversion of a small number of mutations indicated by backcross experiments yielded designs with substitutions to up to 60% of the surface that did support folding and membrane insertion.The β-barrel membrane proteins comprise one of the two structural classes of integral membrane proteins. They are found within the outer membranes of bacteria, mitochondria, and chloroplasts, where they perform a range of structural, transport, and catalytic functions (1). In addition to their biological interest they are increasingly relevant to biotechnology, serving as scaffolds for bacterial surface display (2, 3) and atomically precise pores for nanopore-based DNA sequencing. Although the suitability of natural β-barrel membrane proteins for biotechnology has been improved by protein engineering (3–10), the ability to design membrane proteins de novo would deliver tools customized to meet the demands of each application.De novo design provides a stringent test of our understanding of the determinants of protein folding and stability. Protein design software [e.g., Rosetta (11, 12)] has made tremendous strides in addressing the design problem for small water-soluble proteins (13–15), and design of simplified model α-helical membrane proteins including single transmembrane helices and small bundles (16–20) has also been accomplished. In contrast, a designed β-barrel membrane protein has yet to be reported, perhaps as a consequence of the unique design challenges presented by the folding pathway and architecture of these proteins. Unlike the α-helical membrane proteins, nascent β-barrel membrane proteins must transit the periplasm to the outer membrane, where folding and membrane insertion are thought to occur in concert (21, 22). An extensive network of chaperones maintains the solubility of the unfolded barrel and guides membrane insertion. The C-terminal β-strand is known to interact with the BAM chaperone complex (23–25), which assists the folding of all β-barrel membrane proteins. However, despite recent progress (26–30), we do not fully understand how interactions between chaperones and transiting membrane proteins are directed by sequence-encoded information.Further complicating design is the inside-out architecture of β-barrel membrane proteins. In place of a hydrophobic core is either a central water-filled pore or a solid core composed of polar side chains. The lipid bilayer becomes increasingly hydrophobic at greater depths within the membrane (31), and this environmental anisotropy is reflected in the amino acid composition of the barrel surface. Aliphatic side chains are prevalent toward the center of the membrane, and aromatic side chains are common in the lipid head group regions, where they encircle the barrel in external- and periplasmic-side girdles (32).Recently we developed E_zβ, a membrane depth-dependent, residue-level potential calculated from an ensemble of experimentally determined outer membrane protein structures (33, 34). E_zβ can be used to estimate energetics of membrane insertion to predict transmembrane protein orientation within the bilayer, and to detect oligomerization sites on β-barrel surfaces (34). E_zβ and related statistical functions (35, 36) can recapitulate properties of natural outer membrane proteins (37, 38) and predict the effects of mutations on protein stability and oligomerization (39). Similar potentials have driven computational approaches that have fully redesigned α-helical membrane protein surfaces to convert membrane proteins into water-soluble ones (40–42).Here, we considered whether the complete redesign of the lipid-facing surface of an outer membrane protein using a statistical potential such as E_zβ preserves its structure and function. This approach allowed us to investigate whether membrane insertion requires only a lipid-facing surface composed of depth-appropriate hydrophobic residues, or whether folding requires sequence-specific interstrand interactions, chaperone-recruiting sequences, evolutionarily optimized aromatic girdles, folding nucleation sites, or other design features lost during the population averaging inherent in parameter fitting of statistical potentials.Previous studies have explored the sensitivity of the β-barrel fold and its chaperone recognition mechanisms to mutations. The canonical eight-stranded β-barrel membrane protein OmpA tolerates a limited number of mutations to the lipid-facing surface, provided hydrophobicity is maintained (43, 44). More radically, the eight-stranded barrel OmpX has been duplicated to form a 16-stranded barrel capable of membrane insertion (45). However, the lipid-facing residues of transmembrane β-strands are conserved across homologous β-barrel membrane proteins beyond the extent expected from hydrophobicity alone (46, 47), implying a functional role that has yet to be elucidated.To explore the sequence constraints on β-barrel membrane proteins, we extensively redesigned the lipid-facing surface of E. coli OmpA. We created a series of OmpA variants with entirely or partially redesigned lipid-facing surfaces and tested their ability to insert into the outer membrane of E. coli. Our results indicate that the surfaces of β-barrel membrane proteins are amenable to large-scale redesign, provided that energetically destabilizing substitutions are avoided.     

A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling

Endosome-to-Golgi transport is required for the function of many key membrane proteins and lipids, including signaling receptors, small-molecule transporters, and adhesion proteins. The retromer complex is well-known for its role in cargo sorting and vesicle budding from early endosomes, in most cases leading to cargo fusion with the trans-Golgi network (TGN). Transport from recycling endosomes to the TGN has also been reported, but much less is understood about the molecules that mediate this transport step. Here we provide evidence that the F-BAR domain proteins TOCA-1 and TOCA-2 (Transducer of Cdc42 dependent actin assembly), the small GTPase CDC-42 (Cell division control protein 42), associated polarity proteins PAR-6 (Partitioning defective 6) and PKC-3/atypical protein kinase C, and the WAVE actin nucleation complex mediate the transport of MIG-14/Wls and TGN-38/TGN38 cargo proteins from the recycling endosome to the TGN in Caenorhabditis elegans. Our results indicate that CDC-42, the TOCA proteins, and the WAVE component WVE-1 are enriched on RME-1–positive recycling endosomes in the intestine, unlike retromer components that act on early endosomes. Furthermore, we find that retrograde cargo TGN-38 is trapped in early endosomes after depletion of SNX-3 (a retromer component) but is mainly trapped in recycling endosomes after depletion of CDC-42, indicating that the CDC-42–associated complex functions after retromer in a distinct organelle. Thus, we identify a group of interacting proteins that mediate retrograde recycling, and link these proteins to a poorly understood trafficking step, recycling endosome-to-Golgi transport. We also provide evidence for the physiological importance of this pathway in WNT signaling.Endocytosis mediates the internalization of cell-surface proteins and lipids in small vesicles that bud from the plasma membrane and deliver their cargo to endosomes (1). Once cargo proteins reach the endosomes, one important pathway they may follow is retrograde recycling, in which cargos are delivered from endosomes to the trans-Golgi network (TGN) (2). Many important membrane proteins, including signaling receptors and small molecular transporters, require retrograde recycling (2). Some well-studied examples include the cation-independent mannose 6-phosphate receptor, insulin-stimulated glucose transporter Glut4, and Wls/MIG-14, a protein that ferries Wnt ligands to the cell surface during their secretion (2, 3). Certain toxins and viruses co-opt such retrograde transport pathways during their toxic/infectious cycles. These include the bacterial toxins Shiga and cholera, plant exotoxins ricin and abrin, as well as adeno-associated virus type 5 (AAV5) and HIV-1 (2, 3).Relatively few studies have focused on how such recycling pathways function in polarized epithelial cells, although polarized epithelia are a very abundant and important cell type in the human body. The intestine of the nematode Caenorhabditis elegans is a powerful model system for the study of endocytic recycling in the context of polarized epithelial cells, and can be studied within their normal context of the intact living animal (4, 5). The C. elegans intestine is a simple epithelial tube composed of 20 cells arranged mostly in pairs (6). Like mammalian intestinal epithelial cells, those of the C. elegans intestine display readily apparent apicobasal polarity, with basolateral and apical domains separated by apical junctions (6). The intestinal luminal (apical) membranes display a dense microvillar brush border with an overlying glycocalyx and subapical terminal web (6). The basolateral membrane faces the body cavity, exchanging molecules between the intestine and peripheral tissues.We previously established several model transmembrane cargo markers for the analysis of basolateral endocytosis and recycling in the C. elegans intestine (5, 7–10). These include MIG-14-GFP, hTfR-GFP, and hTAC-GFP (5). MIG-14 (Wntless) and hTfR (human transferrin receptor) enter cells via clathrin-dependent endocytosis (5, 7, 11). hTAC (human IL-2 receptor α-chain) enters cells via clathrin-independent endocytosis (4, 5). hTfR and hTAC recycle to the plasma membrane via recycling endosomes, also known as the endocytic recycling compartment (4, 7). MIG-14 recycles to the TGN, but previous work had not tested whether MIG-14 transits the recycling endosome en route to the Golgi (12–14).Many studies in cultured cell lines indicate that there are multiple routes to the Golgi from endosomes, including proposed routes from the recycling endosome, in addition to the more commonly discussed early endosome and late endosome routes (15–19). For instance, CHO cell pulse–chase analysis of fusion proteins bearing the transmembrane and intracellular domains of retrograde recycling proteins, TGN38 and Furin, showed that TGN38 trafficked from the early endosome to recycling endosome to the Golgi, whereas Furin recycling involved transit from the early endosome to the late endosome to the Golgi (20, 21). TGN38 and Shiga toxin have been shown to require distinct sets of SNARE proteins to complete transport to the Golgi, also indicating that different cargos recycle to the Golgi in different types of vesicles (22). In addition, TGN38 requires recycling endosome regulator Rab11 and its effector FIP1/RCP for retrograde recycling, further indicating that the recycling endosome pathway is important in TGN38 retrieval to the Golgi (23). The recycling endosome regulator and dynamin superfamily-like ATPase EHD1/mRme-1 is also required for transport of several cargos from recycling endosomes to the Golgi (24–26). The cation-independent mannose 6-phosphate receptor has also been reported to require transport through the recycling endosome to reach the TGN (24, 25).Previous whole-genome analysis of genes required for yolk protein endocytosis in the C. elegans oocyte, a process that requires yolk receptor recycling, identified the Rho-family GTPase CDC-42 and its associated proteins PAR-3 and PAR-6 (PDZ domain proteins), as well as the C. elegans homolog of atypical protein kinase C, PKC-3 (27). Together, these proteins are often referred to as the anterior PAR complex, because they function together to establish and maintain anterior–posterior polarity in the early C. elegans embryo (28). This work showed that CDC-42 is enriched on RME-1–positive recycling endosomes in nonpolarized C. elegans coelomocytes and cultured mammalian fibroblasts (27). These and other data implicated the CDC-42/PAR complex in recycling endosome function, but further mechanistic insight was lacking (27, 29, 30). Other work showed that CDC-42–associated Bar-domain proteins TOCA-1 and TOCA-2 function redundantly in yolk endocytosis, also probably functioning at a postendocytic transport step (31).To better understand the function of the TOCA proteins, and potentially the anterior PAR complex, in membrane transport, we set out to analyze their function in the C. elegans intestine using the molecular tools that we had established in this tissue. Unlike the general recycling regulator RME-1, which affects all recycling cargo that we have tested in the C. elegans intestine, we found that toca-1; toca-2 double mutants strongly affected MIG-14 but not hTAC or hTfR. Further analysis connected TOCA-1 and TOCA-2 to the CDC-42/PAR complex in this process, and further indicated that these proteins function with WVE-1, a core subunit of the actin nucleation complex WAVE. These results indicated a requirement for TOCA/CDC-42/PAR/WAVE in retrograde recycling, in addition to the well-known retromer complex, which contains a similar complement of molecules implicated in membrane binding, membrane bending, and actin nucleation. Finally, we established the C. elegans homolog of retrograde recycling cargo TGN38 (TGN-38) as a retrograde recycling cargo in the intestine. Experiments with GFP-tagged TGN-38 allowed us to compare the cargo transport blocks imposed by depletion of a subunit of the retromer complex (SNX-3) with the block imposed by depletion of CDC-42, as a representative of the TOCA/CDC-42/PAR/WAVE group (32). These experiments indicated a specific block at the early endosome after retromer depletion, whereas CDC-42 depletion blocked TGN-38 retrograde recycling most strongly at the recycling endosome. Taken together, these results demonstrate an important role for TOCA/CDC-42/PAR/WAVE acting at the recycling endosome after retromer function at the early endosome. We also provide evidence for the physiological importance of this pathway in WNT signaling, focusing on the polarity of the ALM mechanosensory neurons.