ADP ribosylation factor regulates spectrin binding to the Golgi complex

Homologues of two major components of the well-characterized erythrocyte plasma-membrane-skeleton, spectrin (a not-yet-cloned isoform, βIΣ* spectrin) and ankyrin (Ank_G119 and an ≈195-kDa ankyrin), associate with the Golgi complex. ADP ribosylation factor (ARF) is a small G protein that controls the architecture and dynamics of the Golgi by mechanisms that remain incompletely understood. We find that activated ARF stimulates the in vitro association of βIΣ* spectrin with a Golgi fraction, that the Golgi-associated βIΣ* spectrin contains epitopes characteristic of the βIΣ2 spectrin pleckstrin homology (PH) domain known to bind phosphatidylinositol 4,5-bisphosphate (PtdInsP₂), and that ARF recruits βIΣ* spectrin by inducing increased PtdInsP₂ levels in the Golgi. The stimulation of spectrin binding by ARF is independent of its ability to stimulate phospholipase D or to recruit coat proteins (COP)-I and can be blocked by agents that sequester PtdInsP₂. We postulate that a PH domain within βIΣ* Golgi spectrin binds PtdInsP₂ and acts as a regulated docking site for spectrin on the Golgi. Agents that block the binding of spectrin to the Golgi, either by blocking the PH domain interaction or a constitutive Golgi binding site within spectrin’s membrane association domain I, inhibit the transport of vesicular stomatitis virus G protein from endoplasmic reticulum to the medial compartment of the Golgi complex. Collectively, these results suggest that the Golgi-spectrin skeleton plays a central role in regulating the structure and function of this organelle.     

Phosphoregulatory protein 14-3-3 facilitates SAC1 transport from the endoplasmic reticulum

Most secretory cargo proteins in eukaryotes are synthesized in the endoplasmic reticulum and actively exported in membrane-bound vesicles that are formed by the cytosolic coat protein complex II (COPII). COPII proteins are assisted by a variety of cargo-specific adaptor proteins required for the concentration and export of secretory proteins from the endoplasmic reticulum (ER). Adaptor proteins are key regulators of cargo export, and defects in their function may result in disease phenotypes in mammals. Here we report the role of 14-3-3 proteins as a cytosolic adaptor in mediating SAC1 transport in COPII-coated vesicles. Sac1 is a phosphatidyl inositol-4 phosphate (PI4P) lipid phosphatase that undergoes serum dependent translocation between the endoplasmic reticulum and Golgi complex and controls cellular PI4P lipid levels. We developed a cell-free COPII vesicle budding reaction to examine SAC1 exit from the ER that requires COPII and at least one additional cytosolic factor, the 14-3-3 protein. Recombinant 14-3-3 protein stimulates the packaging of SAC1 into COPII vesicles and the sorting subunit of COPII, Sec24, interacts with 14-3-3. We identified a minimal sorting motif of SAC1 that is important for 14-3-3 binding and which controls SAC1 export from the ER. This LS motif is part of a 7-aa stretch, RLSNTSP, which is similar to the consensus 14-3-3 binding sequence. Homology models, based on the SAC1 structure from yeast, predict this region to be in the exposed exterior of the protein. Our data suggest a model in which the 14-3-3 protein mediates SAC1 traffic from the ER through direct interaction with a sorting signal and COPII.Most of the transmembrane secretory cargo proteins from the endoplasmic reticulum (ER) are selectively exported in cytosolic coat protein complex II (COPII) vesicles via direct interaction of their export motif with the COPII coat. The COPII coat core machinery consists of five cytosolic proteins: Sar1, Sec23, Sec24, Sec13, and Sec31 (secretory pathway proteins) (1). Sec24 is considered to be the primary subunit responsible for binding to membrane cargo proteins at the ER and concentrating them into the forming vesicle (2). Some of these cargo proteins require the assistance of cytosolic or membrane-spanning accessory adaptor proteins for their incorporation into COPII vesicles. Several adaptor proteins have been identified to assist the COPII machinery in yeast (3–5); however, fewer have been characterized in higher eukaryotes. In metazoans, ERGIC-53 mediates the export of blood clotting factors, Cathepsin Z and C and α-1 antitrypsin (6), and SCAP [sterol-regulatory elementary binding protein (SREBP) cleavage activating protein] mediates the regulated transport of SREBP protein from the ER to the Golgi in cells that are sterol-deficient (7). Most COPII adaptor proteins are membrane-embedded, but at least one example of a cytosolic accessory protein, 14-3-3, has been proposed to control the anterograde trafficking of many of cell surface receptor proteins, possibly at the level of the ER (8). 14-3-3s are small (30 kDa), acidic, and ubiquitously expressed eukaryotic proteins that are conserved from yeast to mammals and modulate various cellular processes by interacting with a variety of target proteins (9, 10). These include cell cycle regulation, signaling by MAP kinases, apoptosis, and transfer of signaling molecules between the nucleus and cytosol (11–14). Yeast cell viability depends on the expression of at least one of the two 14-3-3 isoforms (Bmh1 and Bmh2) (15). There are seven different isoforms in mammals (β, γ, δ, ε, η, σ, θ), some of which show differential tissue localization (14). Because of their redundant roles in cellular processes, depleting cellular levels of 14-3-3 to study a particular process poses a challenge. It is thought that their role in trafficking is to interfere with the ER retention/retrieval motif of target membrane proteins, and thus promote the transport of these cargos to the cell surface (16). For some proteins (e.g., KCNK3 and MHC class II, GPR15) (17–19), recruitment of 14-3-3 requires phosphorylation of a residue involved in 14-3-3 binding, whereas in other proteins (e.g., Kir6.2) 14-3-3 recognizes the correct assembly of multimeric proteins (20, 21).In this paper we examine the role of 14-3-3 proteins as an adaptor for COPII vesicular transport of SAC1 (suppressor of actin mutations 1-like protein). SAC1 is a phosphatidyl inositol-4 (PI4) lipid phosphatase that belongs to a family of enzymes with a CX₅R(T/S) Sac catalytic domain, which is conserved from yeast to metazoans. Sac proteins control several cellular processes, including phosphoinositide homeostasis, membrane trafficking, and cytoskeleton organization. SAC1 is a 587-aa transmembrane protein with both N- and C-terminal domains exposed to the cytosol. Deletion of SAC1 in yeast and mammalian cells leads to changes in Golgi morphology and function and a SAC1 mouse knockout is embryonically lethal. Recently, SAC1 has been identified as Drosophila vesicle-associated protein binding partner and down-regulation of Drosophila vesicle-associated protein or SAC1 in Drosophila leads to the pathogenesis associated with amyotrophic lateral sclerosis (22).It has been reported previously that SAC1 is localized to the Golgi membranes only when cells are starved for nutrients or growth factors, but remains in the ER under normal growth conditions (23, 24). Given the role for PI(4)P in vesicle traffic from the trans Golgi network, starvation conditions that lodge SAC1 and thus deplete the local supply of PI(4)P in the Golgi may suppress anterograde traffic in cells that must cease net cell growth. The regulation of SAC1 traffic may be crucial to the control of cell growth and anterograde membrane traffic.The retrieval of mammalian SAC1 from the Golgi to the ER in the presence of growth factors or mitogens is controlled by COPI-mediated retrograde transport and requires the p38 MAPK pathway (23). Although the regulation of SAC1 retrieval from the Golgi has been reported, little is known about the control of SAC1 export from the ER under conditions of serum starvation. Recently, the N-terminal cytoplasmic domain of SAC1 was reported to contribute to Golgi localization in mammalian cells (25). We have established a cell-free reconstitution system that recapitulates the biogenesis and ER export of SAC1 and identified 14-3-3 proteins as an important factor in the packaging of SAC1 into COPII transport vesicles. Given the role of 14-3-3 proteins in various signaling pathways and the fact that SAC1 transport is affected by the p38 MAPK pathway, an understanding of the molecular role of 14-3-3 proteins in vesicular traffic could provide a mechanistic link between signaling and membrane assembly (23).     

Exiting the endoplasmic reticulum

The movement of nascent proteins from sites of synthesis to final cellular or extracellular destinations involves their transport through a distinct series of vesicular compartments. Vesicle biogenesis is regulated by specific proteins and co-factors that control distinct steps including budding, transport, docking, and fusion with target membranes. Budding requires assembly of a coat protein complex on the membrane, membrane deformation and the subsequent cleavage of the nascent vesicle from donor membrane. Coat proteins may also mediate vesicle interactions with the cytoskeleton or insulate the vesicles from fusion with unwanted compartments. Three classes of cytoplasmic coats have been identified. (1) Clathrin, interacting with different adaptor proteins, participates in endocytosis, lysosome biogenesis and as yet unidentified vesicular transport processes that arise in the trans-Golgi region of cells [reviewed in (Kreis, T.E., Lowe, M., Pepperkok, R., 1995. COPs regulating membrane traffic. Ann. Rev. Cell. Dev. Biol. 11, 677--706.)]. (2) The COPI coatomer is involved in retrograde traffic within the Golgi and from the cis-Golgi region to the endoplasmic reticulum (ER). It may also participate in anterograde transport from the ER [reviewed in (Aridor, M., Balch, W.E., 1999. Integration of endoplasmic reticulum signaling in health and disease. Nature 5, 745--751.)]. (3) COPII coats mediate anterograde transport of cargo out of the ER [Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M.F., Ravazazola, M., Amherdt, M., Schekman, R., 1994. COPII: a membrane coat formed by sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell 77, 895--907; Scales, S.J., Gomez, M., Kreis, T.E., 2000. Coat proteins regulating membrane traffic. Int. Rev. Cytol. 195, 67--144.]. The COPII coat is required for budding from the ER and ER to Golgi trafficking. Further, COPII proteins also participate in cargo selection and concentrate some nascent proteins in the budding vesicle. Recent studies have shown that human disease may result from mutations that affect proteins in COPII vesicles.     

Calneurons provide a calcium threshold for trans-Golgi network to plasma membrane trafficking

Phosphatidylinositol 4-OH kinase IIIβ (PI-4Kβ) is involved in the regulated local synthesis of phospholipids that are crucial for trans-Golgi network (TGN)-to-plasma membrane trafficking. In this study, we show that the calcium sensor proteins calneuron-1 and calneuron-2 physically associate with PI-4Kβ, inhibit the enzyme profoundly at resting and low calcium levels, and negatively interfere with Golgi-to-plasma membrane trafficking. At high calcium levels this inhibition is released and PI-4Kβ is activated via a preferential association with neuronal calcium sensor-1 (NCS-1). In accord to its supposed function as a filter for subthreshold Golgi calcium transients, neuronal overexpression of calneuron-1 enlarges the size of the TGN caused by a build-up of vesicle proteins and reduces the number of axonal Piccolo-Bassoon transport vesicles, large dense core vesicles that carry a set of essential proteins for the formation of the presynaptic active zone during development. A corresponding protein knockdown has the opposite effect. The opposing roles of calneurons and NCS-1 provide a molecular switch to decode local calcium transients at the Golgi and impose a calcium threshold for PI-4Kβ activity and vesicle trafficking.     

ER trapping reveals Golgi enzymes continually revisit the ER through a recycling pathway that controls Golgi organization

Whether Golgi enzymes remain localized within the Golgi or constitutively cycle through the endoplasmic reticulum (ER) is unclear, yet is important for understanding Golgi dependence on the ER. Here, we demonstrate that the previously reported inefficient ER trapping of Golgi enzymes in a rapamycin-based assay results from an artifact involving an endogenous ER-localized 13-kD FK506 binding protein (FKBP13) competing with the FKBP12-tagged Golgi enzyme for binding to an FKBP-rapamycin binding domain (FRB)-tagged ER trap. When we express an FKBP12-tagged ER trap and FRB-tagged Golgi enzymes, conditions precluding such competition, the Golgi enzymes completely redistribute to the ER upon rapamycin treatment. A photoactivatable FRB-Golgi enzyme, highlighted only in the Golgi, likewise redistributes to the ER. These data establish Golgi enzymes constitutively cycle through the ER. Using our trapping scheme, we identify roles of rab6a and calcium-independent phospholipase A₂ (iPLA₂) in Golgi enzyme recycling, and show that retrograde transport of Golgi membrane underlies Golgi dispersal during microtubule depolymerization and mitosis.The Golgi apparatus is the major processing and sorting station at the crossroads of the secretory pathway (1–4). It receives newly synthesized proteins from the endoplasmic reticulum (ER), processes them using Golgi-specific enzymes, and sorts them to the plasma membrane (PM) and other final destinations. During this process, specialized sorting and transport machinery of the Golgi filter out selected membrane and protein components, returning them back to the ER for continued use. How the Golgi maintains its structure and function amid this ongoing bidirectional membrane trafficking has been a long-standing debate.A variety of studies have advanced the view that the Golgi apparatus is a dynamic, steady-state system in which resident enzymes continuously recycle back to the ER and return to the Golgi through the same retrograde and anterograde trafficking routes used by other proteins (4–6). This model helps account for the striking dispersal of the Golgi when ER export is blocked (7–9), microtubules are depolymerized (10–12), or cells enter mitosis (13, 14). In each case, Golgi enzymes have been reported to redistribute to the site of transport block in the ER.An alternative model has been proposed that envisions the Golgi as an autonomous organelle with stable components that provide a template for its growth and division. In this model, Golgi enzymes ordinarily remain localized in the Golgi throughout their lifetime and Golgi dispersal under ER export blockade, microtubule depolymerization, or mitotic entry is due to a reversible breakdown of the Golgi itself into smaller elements or vesicles, without contact with the ER or its associated pathways. One primary support for this model comes from studies examining the relationship of Golgi enzymes and ER proteins using a rapamycin-induced protein heterodimerization assay to trap FKBP12- and FKBP-rapamycin binding domain (FRB)-tagged probes in close proximity (15). Using an FRB-tagged, ER-retained version of invariant chain Iip35 (Ii-FRB) (16, 17) and FKBP12-labeled Golgi enzymes, these studies observed little trapping of Golgi enzymes in the ER in the presence of rapamycin (15), including during Golgi dispersal upon mitotic entry (18).If representative of normal Golgi enzyme behavior, the ER trapping assay’s results would call into question the wide range of other observations supporting the steady-state Golgi model and its ER dependency. We thus examined the assay in more detail to determine whether it might be underestimating the extent of Golgi enzyme recycling to the ER. Prior work has shown that an endogenous FK506-binding protein, FKBP13, localizes to the lumen of the ER (19, 20), where, in the presence of rapamycin, it forms a ternary complex with FRB-containing proteins (20). We tested the possibility that binding of endogenous FKBP13 to ER-localized Ii-FRB when rapamycin is introduced might explain why no significant trapping of FKBP12-labeled Golgi enzymes in the ER was observed in the prior work. Consistent with this possibility, we found that coexpressing an FKBP12-tagged ER protein and an FRB-tagged Golgi enzyme marker, conditions where endogenous ER-localized FKBP13 would not sequester the ER trap, resulted in complete redistribution of the Golgi marker into the ER within 4 h of rapamycin treatment. In the ER, FRB-Golgi and FKBP12-ER markers underwent FRET, indicating direct binding upon rapamycin-induced redistribution. These data support the Golgi recycling theory by providing evidence of trapping of Golgi enzymes in the ER.The ability to assess retrograde transport of Golgi enzymes using our modified ER trapping assay enabled us to characterize the pathway while offering additional evidence of its existence. We found that the carriers delivering Golgi enzymes to the ER were tubule-shaped and moved peripherally after extending off from the Golgi. Rab6a and cation-independent phospholipase A₂ (iPLA₂) were required for their delivery to the ER. Golgi enzyme recycling to the ER was also shown to be involved in Golgi fragmentation during microtubule depolymerization and during mitosis. We anticipate that further use of this modified ER trapping assay will provide more insights into the precise nature of the Golgi’s dependence on the ER.     

Rer1p as common machinery for the endoplasmic reticulum localization of membrane proteins

Rer1p, a Golgi membrane protein, is required for the correct localization of an endoplasmic reticulum (ER) membrane protein, Sec12p, by a retrieval mechanism from the cis-Golgi to the ER. To test whether or not the role of Rer1p is common to multiple ER membrane proteins, we examined the localization of two other ER membrane proteins, Sec71p and Sec63p, in the wild-type and rer1 mutant yeast cells, using their fusions with an α-mating factor precursor (Mfα1p). Although Sec71p and Sec63p have completely different topology from Sec12p, their Mfα1p fusion proteins were also mislocalized to the trans-Golgi in the rer1 mutant. Overexpression of these fusions caused their mislocalization to the trans-Golgi even in the wild-type cells, and this mislocalization was partially suppressed by the co-overexpression of Rer1p. Either Sec71p or an artificial chimeric protein whose ER localization depends on Rer1p gave a competitive effect on the localization of the Mfα1-Sec71p fusion, which was abolished in rer1. Thus, Rer1p appears to be one of the common limiting components in the retrieval machinery for ER membrane proteins. The results also suggest that Sec71p and Sec63p depend on ER-Golgi recycling, at least partly, for ER localization. We also examined the effect of a mutation in α-COP, a subunit of yeast coatomer, on the localization of these ER membrane proteins. The Mfα1p fusions of Sec12p, Sec71p, and Sec63p were all more or less mislocalized in ret1–1. These observations imply that the roles of Rer1p and coatomer are much more general than thought before.     

Reversible dissociation of coatomer: Functional characterization of a β/δ-coat protein subcomplex

COPI-coated vesicles mediate protein transport within the early secretory pathway. Their coat consists of ADP ribosylation factor (ARF1, a small guanosine nucleotide binding protein), and coatomer, a cytosolic complex composed of seven subunits, α- to ζ-coat proteins (COPs). For coat formation that initiates budding of a vesicle, ARF1 is recruited to the Golgi membrane from the cytosol in its GTP-bound form, and subsequently, coatomer can bind to the membrane. To identify a minimal structure of coatomer capable to bind to Golgi membranes in an ARF1-dependent manner, we have established a procedure to dissociate coatomer under conditions that allow reassociation of the subunits to a complete and functional complex. After dissociation, subunits or subcomplexes can be isolated and may be expected to be functional. Herein we describe isolation of a subcomplex of coatomer consisting of β- and δ-COPs that is able to bind to Golgi membranes in an ARF1- and GTP-dependent manner.     

Regulation of Golgi structure and secretion by receptor-induced G protein βγ complex translocation

We show that receptor induced G protein βγ subunit translocation from the plasma membrane to the Golgi allows a receptor to initiate fragmentation and regulate secretion. A lung epithelial cell line, A549, was shown to contain an endogenous translocating G protein γ subunit and exhibit receptor-induced Golgi fragmentation. Receptor-induced Golgi fragmentation was inhibited by a shRNA specific to the endogenous translocating γ subunit. A kinase defective protein kinase D and a phospholipase C β inhibitor blocked receptor-induced Golgi fragmentation, suggesting a role for this process in secretion. Consistent with βγ translocation dependence, fragmentation induced by receptor activation was inhibited by a dominant negative nontranslocating γ3. Insulin secretion was shown to be induced by muscarinic receptor activation in a pancreatic β cell line, NIT-1. Induction of insulin secretion was also inhibited by the dominant negative γ3 subunit consistent with the Golgi fragmentation induced by βγ complex translocation playing a role in secretion.     

Cysteine shotgun-mass spectrometry (CS-MS) reveals dynamic sequence of protein structure changes within mutant and stressed cells

Questions of if and when protein structures change within cells pervade biology and include questions of how the cytoskeleton sustains stresses on cells—particularly in mutant versus normal cells. Cysteine shotgun labeling with fluorophores is analyzed here with mass spectrometry of the spectrin–actin membrane skeleton in sheared red blood cell ghosts from normal and diseased mice. Sheared samples are compared to static samples at 37 °C in terms of cell membrane intensity in fluorescence microscopy, separated protein fluorescence, and tryptic peptide modification in liquid chromatography–tandem mass spectrometry (LC-MS/MS). Spectrin labeling proves to be the most sensitive to shear, whereas binding partners ankyrin and actin exhibit shear thresholds in labeling and both the ankyrin-binding membrane protein band 3 and the spectrin–actin stabilizer 4.1R show minimal differential labeling. Cells from 4.1R-null mice differ significantly from normal in the shear-dependent labeling of spectrin, ankyrin, and band 3: Decreased labeling of spectrin reveals less stress on the mutant network as spectrin dissociates from actin. Mapping the stress-dependent labeling kinetics of α- and β-spectrin by LC-MS/MS identifies Cys in these antiparallel chains that are either force-enhanced or force-independent in labeling, with structural analyses indicating the force-enhanced sites are sequestered either in spectrin’s triple-helical domains or in interactions with actin or ankyrin. Shear-sensitive sites identified comprehensively here in both spectrin and ankyrin appear consistent with stress relief through forced unfolding followed by cytoskeletal disruption.     

Phosphatidylserine binding sites in red cell spectrin

Spectrin has been shown to interact with phosphatidylserine (PS), however, the precise binding sites for PS in spectrin have not been defined. In the present study, we have identified specific PS binding sites in spectrin using recombinant spectrin fragments encompassing the entire sequences of both spectrin chains. We show that sites of high affinity are located within eight of the 38 triple-helical structural repeats which make up the bulk of both chains: these are: α8 and α9-10, and β2, β3, β4, β12, β13 and β14, and PS affinity was also found in the non-homologous N-terminal domain of the β-chain. It is noteworthy that the PS-binding sites in β-spectrin are clustered in close proximity to the sites of attachment both of ankyrin and of 4.1R, the proteins engaged in attachment of spectrin to the membrane. We conjecture that direct interaction of spectrin with PS in the membrane complements modulates its interactions with the proteins, and that (considering also the known affinity of 4.1R for PS) the formation of PS-rich lipid domains, which have been observed in the red cell membrane, may be a result.     

Human Papillomavirus L2 Capsid Protein Stabilizes γ-Secretase during Viral Infection

Intracellular trafficking of human papillomavirus (HPV) during virus entry requires γ-secretase, a cellular protease consisting of a complex of four cellular transmembrane (TM) proteins. γ-secretase typically cleaves substrate proteins but it plays a non-canonical role during HPV entry. γ-secretase binds to the HPV minor capsid protein L2 and facilitates its insertion into the endosomal membrane. After insertion, L2 protrudes into the cytoplasm, which allows HPV to bind other cellular factors required for proper virus trafficking into the retrograde transport pathway. Here, we further characterize the interaction between γ-secretase and HPV L2. We show that γ-secretase is required for cytoplasmic protrusion of L2 and that L2 associates strongly with the PS1 catalytic subunit of γ-secretase and stabilizes the γ-secretase complex. Mutational studies revealed that a putative TM domain in HPV16 L2 cannot be replaced by a foreign TM domain, that infectivity of HPV TM mutants is tightly correlated with γ-secretase binding and stabilization, and that the L2 TM domain is required for protrusion of the L2 protein into the cytoplasm. These results provide new insight into the interaction between γ-secretase and L2 and highlight the importance of the native HPV L2 TM domain for proper virus trafficking during entry.     

Identification and characterization of COPIa- and COPIb-type vesicle classes associated with plant and algal Golgi

Coat protein I (COPI) vesicles arise from Golgi cisternae and mediate the recycling of proteins from the Golgi back to the endoplasmic reticulum (ER) and the transport of Golgi resident proteins between cisternae. In vitro studies have produced evidence for two distinct types of COPI vesicles, but the in vivo sites of operation of these vesicles remain to be established. We have used a combination of electron tomography and immunolabeling techniques to examine Golgi stacks and associated vesicles in the cells of the scale-producing alga Scherffelia dubia and Arabidopsis preserved by high-pressure freezing/freeze-substitution methods. Five structurally distinct types of vesicles were distinguished. In Arabidopsis, COPI and COPII vesicle coat proteins as well as vesicle cargo molecules (mannosidase I and sialyltransferase-yellow fluorescent protein) were identified by immunogold labeling. In both organisms, the COPI-type vesicles were further characterized by a combination of six structural criteria: coat architecture, coat thickness, membrane structure, cargo staining, cisternal origin, and spatial distribution. Using this multiparameter structural approach, we can distinguish two types of COPI vesicles, COPIa and COPIb. COPIa vesicles bud exclusively from cis cisternae and occupy the space between cis cisternae and ER export sites, whereas the COPIb vesicles bud exclusively from medial- and trans-Golgi cisternae and are confined to the space around these latter cisternae. We conclude that COPIa vesicle-mediated recycling to the ER occurs only from cis cisternae, that retrograde transport of Golgi resident proteins by COPIb vesicles is limited to medial and trans cisternae, and that diffusion of periGolgi vesicles is restricted.     

The α2δ subunits of voltage-gated calcium channels form GPI-anchored proteins,a posttranslational modification essential for function

Voltage-gated calcium channels are thought to exist in the plasma membrane as heteromeric proteins, in which the α1 subunit is associated with two auxiliary subunits, the intracellular β subunit and the α₂δ subunit; both of these subunits influence the trafficking and properties of Ca_V1 and Ca_V2 channels. The α₂δ subunits have been described as type I transmembrane proteins, because they have an N-terminal signal peptide and a C-terminal hydrophobic and potentially transmembrane region. However, because they have very short C-terminal cytoplasmic domains, we hypothesized that the α₂δ proteins might be associated with the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor attached to δ rather than a transmembrane domain. Here, we provide biochemical, immunocytochemical, and mutational evidence to show that all of the α₂δ subunits studied, α₂δ-1, α₂δ-2, and α₂δ-3, show all of the properties expected of GPI-anchored proteins, both when heterologously expressed and in native tissues. They are substrates for prokaryotic phosphatidylinositol-phospholipase C (PI-PLC) and trypanosomal GPI-PLC, which release the α₂δ proteins from membranes and intact cells and expose a cross-reacting determinant epitope. PI-PLC does not affect control transmembrane or membrane-associated proteins. Furthermore, mutation of the predicted GPI-anchor sites markedly reduced plasma membrane and detergent-resistant membrane localization of α₂δ subunits. We also show that GPI anchoring of α₂δ subunits is necessary for their function to enhance calcium currents, and PI-PLC treatment only reduces calcium current density when α₂δ subunits are coexpressed. In conclusion, this study redefines our understanding of α₂δ subunits, both in terms of their role in calcium-channel function and other roles in synaptogenesis.     

βIV-Spectrin and CaMKII facilitate Kir6.2 regulation in pancreatic beta cells

Identified over a dozen years ago in the brain and pancreatic islet, β_IV-spectrin is critical for the local organization of protein complexes throughout the nervous system. β_IV-Spectrin targets ion channels and adapter proteins to axon initial segments and nodes of Ranvier in neurons, and β_IV-spectrin dysfunction underlies ataxia and early death in mice. Despite advances in β_IV-spectrin research in the nervous system, its role in pancreatic islet biology is unknown. Here, we report that β_IV-spectrin serves as a multifunctional structural and signaling platform in the pancreatic islet. We report that β_IV-spectrin directly associates with and targets the calcium/calmodulin-dependent protein kinase II (CaMKII) in pancreatic islets. In parallel, β_IV-spectrin targets ankyrin-B and the ATP-sensitive potassium channel. Consistent with these findings, β_IV-spectrin mutant mice lacking CaMKII- or ankyrin-binding motifs display selective loss of expression and targeting of key protein components, including CaMKIIδ. β_IV-Spectrin–targeted CaMKII directly phosphorylates the inwardly-rectifying potassium channel, Kir6.2 (alpha subunit of K_ATP channel complex), and we identify the specific residue, Kir6.2 T224, responsible for CaMKII-dependent regulation of K_ATP channel function. CaMKII-dependent phosphorylation alters channel regulation resulting in K_ATP channel inhibition, a cellular phenotype consistent with aberrant insulin regulation. Finally, we demonstrate aberrant K_ATP channel phosphorylation in β_IV-spectrin mutant mice. In summary, our findings establish a broader role for β_IV-spectrin in regulation of cell membrane excitability in the pancreatic islet, define the pathway for CaMKII local control in pancreatic beta cells, and identify the mechanism for CaMKII-dependent regulation of K_ATP channels.The coordinate expression, localization, and regulation of ion channels are critical for excitable cell biology, namely, neuronal transmission, cardiac excitation–contraction coupling, endocrine/exocrine secretory regulation, and skeletal muscle function. The spectrin family of polypeptides, originally discovered as a critical component of the erythrocyte plasma membrane over 30 y ago, is now known to play important structural roles in both nonexcitable and excitable cells (1, 2). Spectrin alpha (α) and beta (β) subunits form antiparallel dimers that self-associate to form tetramers, creating a submembranous scaffolding network anchored to actin filaments via β-spectrin (2). Whereas two genes encode α-spectrin polypeptides (α_I-, α_II-spectrin), spectrin family diversity is created through assembly of α-spectrin products with five β-spectrin genes (encoding β_I–β_V-spectrin) (2). Over a dozen years ago, β_IV-spectrin was identified in brain and pancreatic islets (3). Since these findings, a host of studies have identified β_IV-spectrin as a critical player for defining local membrane domains in the nervous system (4–6). Notably, β_IV-spectrin complexes serve essential roles in targeting ankyrin and associated membrane proteins, to axon initial segments and nodes of Ranvier. Dysfunction in β_IV-spectrin leads to aberrant membrane protein targeting, cell and animal phenotypes, and early death (6, 7). Despite these striking findings in brain, the role of β_IV-spectrin in pancreatic islets is unknown.Here, we define a role for β_IV-spectrin in regulating membrane excitability in islets. β_IV-spectrin directly associates with ankyrin-B and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). We define the structural requirements for these interactions and show that β_IV-spectrin associates with additional islet proteins, including actin and the ATP sensitive potassium channel (K_ATP channel). Notably, β_IV-spectrin mutant mice that lack ankyrin- or CaMKII-binding activity display coordinate loss of ankyrin, K_ATP channel, and CaMKII expression and targeting. Finally, we demonstrate that β_IV-spectrin is critical for CaMKII-dependent regulation of the K_ATP channel through Kir6.2 residue T224 and show that Kir6.2 T224 phosphorylation is altered in β_IV-spectrin mutant mice. In summary, our findings provide initial insight into the role of β_IV-spectrin in the expression, targeting, and regulation of critical membrane and signaling proteins in islets.     

Structural basis for the binding of tryptophan-based motifs by δ-COP

Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding βγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αβ’ε-COP B-subcomplex. We present the structure of the C-terminal μ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP μ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wx_n(1–6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing.COPI vesicles mediate retrograde trafficking from the Golgi to the endoplasmic reticulum (ER) and within the Golgi (reviewed in refs. 1–3). The COPI vesicle coat consists of two major components, coatomer and the small GTPase ADP ribosylation factor 1 (Arf1). Coatomer is an ∼600 kDa heteroheptameric complex consisting of two linked subcomplexes, the βγδζ-COP F-subcomplex and the αβ’ε-COP B-subcomplex, all conserved from yeast to humans.Recent studies have led to a model for COPI-coated vesicle formation in which the F-subcomplex functions as a traditional Arf1:GTP effector but with two membrane-attached Arf1:GTP molecules binding to quasi-equivalent sites on a single F-subcomplex, recruiting F-subcomplex and its associated B-subcomplex onto the membrane en bloc (4). Once the vesicle has budded from its donor membrane, an Arf GTPase-activating protein (ArfGAP) catalyzes the hydrolysis of GTP on Arf1, and the Arf1:GDP dissociates from the membrane, followed later by the dissociation of the coatomer complex that was bound to the transport vesicle (5). ArfGAPs also have been proposed to function at different stages in vesicle biogenesis, both as terminators and, during an earlier cargo-editing step, as effectors (reviewed in depth in ref. 6). In the final stages of its life, a COPI-coated vesicle docks with the ER via the Dsl1-tethering complex, completes uncoating, and finally undergoes SNARE-mediated fusion with the ER (7–9).The two main ArfGAPs associated with COPI-dependent retrograde transport in yeast are Gcs1p and Glo3p. These proteins can substitute for one another, at least partially, in COPI vesicle-mediated transport, although Glo3p, which binds the γ-COP appendage domain, has been proposed to play the more significant role (10–12). Cassel and coworkers (13) have demonstrated, for mammalian ArfGAP1, that a di-tryptophan motif (WxxxxW) located within the unstructured C terminus of the protein can bind to the mammalian δ-COP C-terminal μ-homology domain (MHD). Intriguingly, this tryptophan motif resembles a proposed yeast δ-COP–binding motif termed “δL,” which was identified by yeast two-hybrid screening and then discovered in a cytosolic protein which at that time was of uncertain function in COPI vesicle trafficking (14). Subsequently similar motifs were identified in the central low complexity region (the so-called “lasso”) of the Dsl1p subunit of the yeast Dsl1 tethering complex (7–9, 15). Here we reveal the structure of Saccharomyces cerevisiae δ-COP MHD and the basis for its binding to di-tryptophan–based motifs from Dsl1p and also demonstrate that a related WxxF motif from the extreme C terminus of Gcs1p binds at the same site on δ-COP. The functional relevance of this binding site on δ-COP is indicated by the observation that mutations in the site that abolish motif binding can affect CPY trafficking/processing and cause growth phenotypes.     

Brefeldin A-inhibited guanine nucleotide-exchange activity of Sec7 domain from yeast Sec7 with yeast and mammalian ADP ribosylation factors

The Saccharomyces cerevisiae Sec7 protein (ySec7p), which is an important component of the yeast secretory pathway, contains a sequence of ≈200 amino acids referred to as a Sec7 domain. Similar Sec7 domain sequences have been recognized in several guanine nucleotide-exchange proteins (GEPs) for ADP ribosylation factors (ARFs). ARFs are ≈20-kDa GTPases that regulate intracellular vesicular membrane trafficking and activate phospholipase D. GEPs activate ARFs by catalyzing the replacement of bound GDP with GTP. We, therefore, undertook to determine whether a Sec7 domain itself could catalyze nucleotide exchange on ARF and found that it exhibited brefeldin A (BFA)-inhibitable ARF GEP activity. BFA is known to inhibit ARF GEP activity in Golgi membranes, thereby causing reversible apparent dissolution of the Golgi complex in many cells. The His₆-tagged Sec7 domain from ySec7p (rySec7d) synthesized in Escherichia coli enhanced binding of guanosine 5′-[γ-[³⁵S]thio]triphosphate by recombinant yeast ARF1 (ryARF1) and ryARF2 but not by ryARF3. The effects of rySec7d on ryARF2 were inhibited by BFA in a concentration-dependent manner but not by inactive analogues of BFA (B-17, B-27, and B-36). rySec7d also promoted BFA-sensitive guanosine 5′-[γ-thio]triphosphate binding by nonmyristoylated recombinant human ARF1 (rhARF1), rhARF5, and rhARF6, although the effect on rhARF6 was very small. These results are consistent with the conclusion that the yeast Sec7 domain itself contains the elements necessary for ARF GEP activity and its inhibition by BFA.     

Golgi and plasma membrane pools of PI(4)P contribute to plasma membrane PI(4,5)P2 and maintenance of KCNQ2/3 ion channel current

Plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] regulates the activity of many ion channels and other membrane-associated proteins. To determine precursor sources of the PM PI(4,5)P₂ pool in tsA-201 cells, we monitored KCNQ2/3 channel currents and translocation of PH_PLCδ1 domains as real-time indicators of PM PI(4,5)P₂, and translocation of PH_OSH2×2, and PH_OSH1 domains as indicators of PM and Golgi phosphatidylinositol 4-phosphate [PI(4)P], respectively. We selectively depleted PI(4)P pools at the PM, Golgi, or both using the rapamycin-recruitable lipid 4-phosphatases. Depleting PI(4)P at the PM with a recruitable 4-phosphatase (Sac1) results in a decrease of PI(4,5)P₂ measured by electrical or optical indicators. Depleting PI(4)P at the Golgi with the 4-phosphatase or disrupting membrane-transporting motors induces a decline in PM PI(4,5)P₂. Depleting PI(4)P simultaneously at both the Golgi and the PM induces a larger decrease of PI(4,5)P₂. The decline of PI(4,5)P₂ following 4-phosphatase recruitment takes 1–2 min. Recruiting the endoplasmic reticulum (ER) toward the Golgi membranes mimics the effects of depleting PI(4)P at the Golgi, apparently due to the trans actions of endogenous ER Sac1. Thus, maintenance of the PM pool of PI(4,5)P₂ appears to depend on precursor pools of PI(4)P both in the PM and in the Golgi. The decrease in PM PI(4,5)P₂ when Sac1 is recruited to the Golgi suggests that the Golgi contribution is ongoing and that PI(4,5)P₂ production may be coupled to important cell biological processes such as membrane trafficking or lipid transfer activity.This paper concerns the dynamics of cellular pools of phosphoinositides, a family of phospholipids located on the cytoplasmic leaflet of cellular membranes, that maintain cell structure, cell motility, membrane identity, and membrane trafficking; they also play key roles in signal transduction (1). Phosphatidylinositol (PI) can be phosphorylated at three positions to generate seven additional species. The subcellular localization of each phosphoinositide is tightly governed by the concurrent presence of lipid kinases and lipid phosphatases (2, 3), giving each membrane within the cell a unique and dynamic phosphoinositide signature (4). Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is localized to the inner leaflet of the plasma membrane (PM) and is the major substrate of phospholipase C (PLC). As a consequence, PI(4,5)P₂ levels are dynamically regulated by G_q-coupled receptors activating PLC. The activity of lipid kinases and phosphatases also can be modulated by signaling; for example, a PI 4-kinase, when associated with neuronal calcium sensor-1, is accelerated in response to elevated calcium that occurs with PI(4,5)P₂ cleavage (5). In addition, transient apposition between organelles can alter phosphoinositide levels by presenting membrane-bound phosphatases in trans. For example, the endoplasmic reticulum (ER) can make contacts with the Golgi, allowing 4-phosphatases of the ER to dephosphorylate Golgi phosphatidylinositol 4-phosphate [PI(4)P] (6–8).PI(4,5)P₂ is a dynamically regulated positive cofactor required for the activity of many plasma membrane ion channels (9). Current in KCNQ2/3 channels (the molecular correlate of neuronal M-current) can be turned off in a few seconds by depletion of PI(4,5)P₂ following activation of PLC through G_q-coupled M₁ muscarinic receptors (M₁Rs) (10–12). Given the importance of PI(4,5)P₂, we wanted to understand better how it is sourced from its precursor PI(4)P within the cell. How do subcellular compartments influence PI(4,5)P₂ abundance at the plasma membrane? PI(4,5)P₂ is derived from PI in two steps: PI 4-kinases make PI(4)P, and PI(4)P 5-kinases make PI(4,5)P₂. Thus, PI(4)P is the immediate precursor of PI(4,5)P₂. Mammalian cells express at least four distinct isoforms of PI 4-kinase that phosphorylate PI on the 4 position to generate PI(4)P and are commonly referred to as PI4K types II (α and β) and III (α and β) (1, 13). PI 4-kinase type IIIα generates PI(4)P at both the Golgi and the PM (14–16). Originally thought to be localized to an ER/Golgi compartment (17, 18), recent experiments show that it is targeted to the plasma membrane by a palmitoylated peripheral membrane protein (16). PI 4-kinase IIIβ is said to be localized to the Golgi and nucleus and contributes to the biosynthesis of Golgi PI(4)P through its association with Arf1 and neuronal calcium sensor 1 (19–21). Inhibition of PI 4-kinase IIIα and -β with micromolar concentrations of wortmannin prevents the replenishment of PM PI(4,5)P₂ following PLC activation (10, 22, 23). Type IIα and IIβ PI4Ks are membrane-bound proteins due to the palmitoylation of a conserved stretch of cysteines in their catalytic domains (24). Immunocytochemical analysis has revealed that they are mostly associated with trans-Golgi, endoplasmic reticulum, and endosomal membranes (25–27). These type IIα and IIβ enzymes are blocked by adenosine and calcium, but are resistant to wortmannin. Therefore, they are not thought to contribute to the recovery of PM PI(4,5)P₂ following G_q-receptor activation (10, 24). Our understanding of the contribution of the PI 4-kinase isoforms is undergoing refinement by accumulating information concerning the unique localization, trafficking, and activity of each PI 4-kinase.Although PI 4-kinase isoforms are present in the membranes of several organelles, the most abundant pools of PI(4)P appear to be those of the PM, Golgi, and secretory vesicles (13–15, 28). A need for Golgi PI(4)P in the maintenance of PM PI(4,5)P₂ was indirectly revealed when plasma membrane PI(4,5)P₂ recovery was slowed following recruitment of a 4-phosphatase to the trans-Golgi network (28). Depletion of PM PI(4)P has been shown to result in small changes to PM PI(4,5)P₂ (15, 22), and knockout of the PM-bound PI 4-kinase IIIα resulted in loss of PI(4)P and a relocation of PI(4,5)P₂ biosensors to intracellular membranes (16). Nevertheless, others have proposed that PM PI(4)P is redundant for the synthesis of PM PI(4,5)P₂ (29–31) and may not serve as its immediate precursor because treatment with the type III PI 4-kinase inhibitor wortmannin or recruiting a 4-phosphatase to the PM had little effect on the PM localization of the PI(4,5)P₂ reporter, the pleckstrin homology (PH) domain from PLCδ1 (PH_PLCδ1).Here, we revisit the relative contributions of PI(4)P pools to PM PI(4,5)P₂. We find that the majority of PM PI(4,5)P₂ needed for maintenance of KCNQ currents comes from two precursor pools of PI(4)P in the cell, one in the PM and the other in the Golgi. The PM pool makes the larger contribution, but the contribution from both locations is significant and ongoing.