Sequences in the N-terminal cytoplasmic domain of Saccharomyces cerevisiae maltose permease are required for vacuolar degradation but not glucose-induced internalization

In Saccharomyces cerevisiae, glucose addition to maltose fermenting cells causes a rapid loss of maltose transport activity and ubiquitin-mediated vacuolar proteolysis of maltose permease. GFP-tagged Mal61 maltose permease was used to explore the role of the N-terminal cytoplasmic domain in glucose-induced inactivation. In maltose-grown cells, Mal61/HA-GFP localizes to the cell surface and, surprisingly, to the vacuole. Studies of end3Δ and doa4Δ mutants indicate that a slow constitutive internalization of Mal61/HA-GFP is required for its vacuolar localization. Site-specific mutagenesis of multiple serine/threonine residues in a putative PEST sequence of the N-terminal cytoplasmic domain of maltose permease blocks glucose-induced Mal61p degradation but does not affect the rapid loss of maltose transport activity associated with glucose-induced internalization. The internalized multiple Ser/Thr mutant protein co-localizes with Snf7p in a putative late endosome or E-compartment. Further, alteration of a putative dileucine [D/EExxxLL/I] motif at residues 64–70 causes a significant defect in maltose transport activity and mislocalization to an E-compartment but appears to have little impact on glucose-induced internalization. We conclude that the N-terminal cytoplasmic domain of maltose permease is not the target of the signaling pathways leading to glucose-induced internalization of Mal61 permease but is required for its subsequent delivery to the vacuole for degradation.     

Structure of the knob protein (KP) gene of Plasmodium falciparum

We have determined the nucleotide sequence of the gene encoding the knob protein (KP) of Plasmodium falciparum (FCR-3/Gambia). The gene is interrupted by an intron which contains 34 imperfect tandemly repeated ATTTT sequences. The first exon encodes 33 amino acids with a hydrophobic core typical of signal peptides. The second exon has an open translational reading frame for 597 amino acids. The deduced protein sequence indicates that KP has multiple structural domains; unlike the N-terminal histidine-rich domain which we described previously, the C-terminal half is rich in lysine residues. Consistent with the apparent association of KP with the cytoplasmic surface of the host erythrocyte membrane, the protein is highly charged and hydrophilic.     

The C-terminal domain is essential for protective activity of the Bordetella pertussis adenylate cyclase-hemolysin.

The adenylate cyclase-hemolysin of Bordetella pertussis consists of a cell-invasive N-terminal adenylate cyclase domain linked to a C-terminal RTX hemolysin containing extensive glycine-rich repeats. The toxin is an essential virulence factor required in the initial stages of infection. Adenylate cyclase-hemolysin was also shown to be a potent vaccinating antigen inducing protection against B. pertussis colonization of the mouse respiratory tract. This protective activity depends on a posttranslational fatty-acylation modification. We used a set of deletion derivatives of the recombinant adenylate cyclase-hemolysin to localize the protective epitopes on the 1,706-residue toxin. We show that specific anti-adenylate cyclase-hemolysin antibodies present in the sera of B. pertussis-infected mice and humans are directed predominantly against the modification-and-repeat portion of the toxin, contained in the last 800 residues of the adenylate cyclase-hemolysin. These antibodies appear to recognize conformational epitopes present only in a structure formed by the intact C-terminal half of the toxin. There was no correlation between the capacity of the truncated adenylate cyclase-hemolysin derivatives to induce both toxin-neutralizing antibodies upon immunization of mice and protective immunity. However, only the truncated proteins which were recognized by the sera of infected mice and humans and which had their last 800 residues intact had the capacity to induce protection of mice against colonization by B. pertussis. This indicates that the structure of the modification-and-repeat region of adenylate cyclase-hemolysin is critical for its protective activity.     

Coronavirus-induced membrane fusion requires the cysteine-rich domain in the spike protein

The spike glycoprotein of mouse hepatitis virus strain A59 mediates the early events leading to infection of cells, including fusion of the viral and cellular membranes. The spike is a type I membrane glycoprotein that possesses a conserved transmembrane anchor and an unusual cysteine-rich (cys) domain that bridges the putative junction of the anchor and the cytoplasmic tail. In this study, we examined the role of these carboxyl-terminal domains in spike-mediated membrane fusion. We show that the cytoplasmic tail is not required for fusion but has the capacity to enhance membrane fusion activity. Chimeric spike protein mutants containing substitutions of the entire transmembrane anchor and cys domain with the herpes simplex virus type 1 glycoprotein D (gD-1) anchor demonstrated that fusion activity requires the presence of the A59 membrane-spanning domain and the portion of the cys domain that lies upstream of the cytoplasmic tail. The cys domain is a required element since its deletion from the wild-type spike protein abrogates fusion activity. However, addition of the cys domain to fusion-defective chimeric proteins was unable to restore fusion activity. Thus, the cys domain is necessary but is not sufficient to complement the gD-1 anchor and allow for membrane fusion. Site-specific mutations of conserved cysteine residues in the cys domain markedly reduce membrane fusion, which further supports the conclusion that this region is crucial for spike function. The results indicate that the carboxyl-terminus of the spike transmembrane anchor contains at least two distinct domains, both of which are necessary for full membrane fusion.     

Sequence requirements for the nuclear localization of the murine cytomegalovirus M44 gene product pp50.

The murine cytomegalovirus (MCMV) M44 gene product pp50 is normally present in the nuclei of virus-infected cells. During transient expression of pp50 in COS-1 cells, the phosphoprotein was readily detectable in the nuclei, indicating that it possesses a nuclear localization signal (NLS). Studies on the subcellular locations of N- and C-terminal deletion mutants of pp50 suggested that alterations in both the C terminus and the highly conserved N-terminal domains of pp50 affect nuclear localization. In particular, the C-terminal 11 amino acids of pp50, which includes a "KKQK" motif, were able to mediate the import of a beta-galactosidase fusion protein into the nucleus. The pair of lysine residues in this motif constitutes an essential element of the C-terminal NLS as mutation of this motif to AAQK directly affected the nuclear localization of either pp50 or beta-galactosidase fusion proteins containing the C-terminal portion of pp50. Furthermore our results indicated that the functionality of the C-terminal NLS is dependent on the structural integrity of the highly conserved N-terminal portion of the molecule, as deletion of amino acids 157-201 alone adversely affected nuclear localization. In the absence of a functional C-terminal NLS, the subcellular localization of pp50 is sensitive to potential conformational changes induced by mutations within the N-terminal half of the molecule. Under those circumstances, mutation of the YK residues at position 22-23 or deletion of amino acids 267-283 was sufficient to produce a protein that was impaired in nuclear import or retention.     

Mapping of novel autoreactive epitopes of the diabetes-associated autoantigen IA-2

IA-2, a member of the tyrosine phosphatase family, has been identified as a dominant autoantigen in type 1 diabetes. To define humoral IA-2 epitopes, we generated a panel of IA-2 deletion mutants and chimeric proteins using the highly homologous tyrosine phosphatase-like protein IA-2beta. Analysis of autoantibody reactivity in 111 IA-2 antibody positive sera from patients with type 1 diabetes revealed that humoral epitopes cluster to several domains of the intracytoplasmic part of IA-2 [IA-2ic, amino acid (aa) 604-979]. Immunodominant epitopes were found in the first N-terminal 73 amino acids (56% positive), in the middle domain residing between residues 699-874 (45% positive) and the C-terminus depending on the presence of aa 931-979 (at least 37% positive). Competition experiments with overlapping peptides revealed that autoantibody binding towards the N-terminus was dependent on residues 621-628. In the C-terminal domain, two novel conformation-dependent epitopes were identified. The first epitope requires the presence of the C-terminal part of IA-2 (aa 933-979) and an IA-2-specific region between residues 771-932. Reactivity against the second epitope was dependent on intact C-terminal domains as well as residues in the middle (aa 887-932) and N-terminal regions (aa 604-771) which are conserved in IA-2 and IA-2beta. We here defined novel autoantigenic determinants in the N-terminus of IA-2 and characterized conformational epitopes residing in the C-terminal region or spanning from C-terminal residues to the N-terminal domain of IA-2ic. The identification of dominant target regions of diabetes-specific autoantibodies may help to elucidate the molecular mechanisms involved in the autoimmunity towards IA-2.     

The C terminus of YopT is crucial for activity and the N terminus is crucial for substrate binding

Recently, it was shown that Yersinia outer protein T (YopT) belongs to a new family of cysteine proteases containing invariant C, H, and D residues that are crucial for its activity. YopT cleaves RhoA, Rac, and Cdc42 at their C termini, thereby releasing them from the membrane. Moreover, YopT inhibits the Rho-rhotekin and Rho-guanine nucleotide dissociation inhibitor interactions. To characterize the active domain of YopT, we constructed N- and C-terminal truncations and expressed them as glutathione S-transferase fusion proteins in Escherichia coli. The toxin fragments were tested for stability by trypsin digestion. The activity of the proteins was studied by membrane release assay, rhotekin pulldown experiments, and microinjection. Whereas deletion of the first 74 N-terminal amino acids did not influence the activity of YopT, deletion of 8 amino acids from the C terminus led to complete loss of activity. N-terminal deletion of 100 amino acids led to an inactive protein, although it still contained the amino acids C139, H258, and D274, which are essential for catalysis. Loss of activity of the N-terminal deletions corresponded to the block of interaction with RhoA, indicating that residues 75 to 100 of YopT are essential for binding to the GTPase. By contrast, when up to 15 amino acids of the C terminus were deleted, the protein had no activity but was still able to interact with RhoA, suggesting a role for the C terminus in the enzyme activity of YopT.     

An oligosaccharide at the C-terminus of the F-specific domain in the stalk of the human parainfluenza virus 3 hemagglutinin-neuraminidase modulates fusion

The promotion of membrane fusion by the fusion (F) protein of human parainfluenza virus 3 (hPIV3) is dependent on a virus-specific contribution from the hemagglutinin-neuraminidase (HN) protein. By evaluation of chimeric hPIV3-Newcastle disease virus (NDV) HN proteins, we have previously shown that hPIV3-F-specificity is determined by a domain that extends from the middle of the membrane anchor to the 82nd residue in the ectodomain [Virology 209, (1995) 457; Arch. Virol. 13 (1997) 115]. If the corresponding NDV-derived residues replace the two C-terminal residues in this domain, no fusion is detected. However, these substitutions restore a glycosylation site present in NDV HN, but not in hPIV3 HN. Deletion of this site from a nested set of chimeras with hPIV3-derived N-terminal portions of decreasing length partially restores fusion, suggesting that an oligosaccharide near the top of hPIV3 HN stalk modulates fusion. In addition, further mutational analyses show that a chimera with only 125 N-terminal hPIV3-derived residues (72 in the stalk) actually promotes fusion more efficiently than the wt protein. These findings localize the C-terminus of the F-specific domain in hPIV3 HN a full 10 residues closer to the membrane than previously shown.     

Impaired interaction of naturally occurring mutant NF2 protein with actin-based cytoskeleton and membrane

Although schwannomin, the product of the neurofibromatosis type 2 gene, shares homology with three cytoskeleton-to-membrane protein linkers defining the ERM family, the mechanism by which it exerts a tumor suppressive activity remains elusive. Based on the knowledge of naturally occurring mutations, a functional study of schwannomin was initiated. Constructs encoding the two wild-type isoforms and nine mutant forms were transfected into HeLa cells. Transiently expressed wild-type isoforms were both observed underneath the plasma membrane. At this location they were detergent insoluble and redistributed by a cytochalasin D treatment, suggesting interaction with actin-based cytoskeletal structures. Proteins with single amino acid substitutions at positions 219 and 220 demonstrated identical properties. Three different truncated schwannomins, that are prototypic for most naturally occurring NF2 mutations, were affected neither in their location nor in their cytochalasin D sensitivity. However, they were revealed to be detergent soluble, indicating a relaxed interaction with the actin-based structures. An increased solubility was also observed for a mutant with a single amino acid substitution at position 360 in the C-terminal half of the protein. Mutant proteins with either a single amino acid deletion at position 118 or an 83 amino acid deletion within the N-terminal domain had lost the submembraneous localization and tended to accumulate in perinuclear patches that were unaffected by cytochalasin D treatment. A similar behavior was observed when the N- terminal domain was entirely deleted. Taken together these observations suggest that the N-terminal domain is the main determinant that localizes the protein at the membrane where it interacts weakly with actin-based cytoskeletal structures. The C-terminal domain potentiates this interaction. With rare exceptions, most naturally occurring mutant schwannomins that have lost their tumor suppressive activity are impaired in an interaction involving actin-based structures and are no longer firmly maintained at the membrane.      

Role of predicted transmembrane domains for type III translocation, pore formation, and signaling by the Yersinia pseudotuberculosis YopB protein

YopB is a 401-amino-acid protein that is secreted by a plasmid-encoded type III secretion system in pathogenic Yersinia species. YopB is required for Yersinia spp. to translocate across the host plasma membrane a set of secreted effector proteins that function to counteract immune signaling responses and to induce apoptosis. YopB contains two predicted transmembrane helices (residues 166 to 188 and 228 to 250) that are thought to insert into the host plasma membrane during translocation. YopB is also required for pore formation and host-cell-signaling responses to the type III machinery, and these functions of YopB may also require membrane insertion. To elucidate the importance of membrane insertion for YopB function, YopB proteins containing helix-disrupting double consecutive proline substitutions in the center of each transmembrane domain were constructed. Yersinia pseudotuberculosis strains expressing the mutant YopB proteins were used to infect macrophages or epithelial cells. Effector translocation, pore formation, and host-cell-signaling responses were studied. Introduction of helix-disrupting substitutions into the second transmembrane domain of YopB resulted in a nonfunctional protein that was not secreted by the type III machinery. Introduction of helix-disrupting substitutions into the first transmembrane domain of YopB resulted in a protein that was fully functional for secretion and for interaction with YopD, another component of the translocation machinery. However, the YopB protein with helix-disrupting substitutions in the first transmembrane domain was partially defective for translocation, pore formation, and signaling, suggesting that all three functions of YopB involve insertion into host membrane.     

Major histocompatibility complex-linked transport proteins and antigen processing

The major histocompatibility complexes of mice, rats and humans each contain a pair of related genes, Tap-1 and Tap-2, that encode members of a large superfamily of proteins having similar structure and function. The TAP-1 (previously called HAM1 in the mouse) and TAP-2 (HAM2) proteins each contain 6-8 predicted membrane-spanning alpha helices, and a cytoplasmic domain containing a putative ATP-binding site. Recent evidence suggests that a functional TAP-1/TAP-2 heterodimer is required for efficient presentation of antigens to CD8+ cytotoxic T cells. This heterodimer resides in the membrane of the endoplasmic reticulum (ER), and probably functions to transport peptides (produced in the cytoplasm) into the ER lumen for binding to MHC class I molecules.     

ZZ domain is essentially required for the physiological binding of dystrophin and utrophin to beta-dystroglycan

An intracellular protein, dystrophin, plays an important role in keeping muscle fibers intact by binding at its N-terminal end to the subsarcolemmal cytoskeletal actin network and via its C-terminal end to the transmembraneous protein beta-dystroglycan. Duchenne muscular dystrophy is caused by the loss of dystrophin, which can result from the loss of this binding. The N-terminal part of the latter binding site of dystrophin has been well documented using overlay assay and X-ray diffraction assays. However, the binding site at the C-terminal region of dystrophin has not been examined in detail. In the present work, we report a detailed analysis of the C-terminal binding domain as follows. (1). The full binding activity corresponding to the effective binding in vivo is expressed by the dystrophin fragment spanning amino acids 3026-3345 containing the ZZ domain at the C-terminus. Determination of this binding range is important not only for understanding of the mechanism of dystrophy, but also useful for the design of truncated dystrophin constructs for gene therapy. (2). The ZZ domain binds to EF1 domain in the dystrophin fragment to reinforce the binding activity. (3). The cysteine 3340 in the ZZ domain is essential for the binding of dystrophin to beta-dystroglycan. A reported case of DMD due to missense mutation C3340Y may be caused by inability to fix dystrophin beneath the cell membrane. (4). The binding mode of utrophin is different from that of dystrophin. The difference is conspicuous concerning the cysteine residues present in the ZZ domain.     

Analysis of the tyrosine phosphorylation and calcium fluxing of human CD6 isoforms with different cytoplasmatic domains

CD6 is a cell surface glycoprotein that functions both as a co-stimulatory and adhesion receptor on T cells. Recently we have described CD6 isoforms (CD6a, b, c, d, e) that arise via alternative splicing of exons encoding the cytoplasmic region of the molecule. CD6 becomes phosphorylated on tyrosine (Tyr) residues following stimulation through the T cell receptor (TCR) complex. Since the phosphorylation of Tyr residues renders some cell surface receptors competent for interactions with proteins of intracellular signaling pathways, we wanted to determine which region(s) and residues in the cytoplasmic domain of CD6 were important for phosphorylation on Tyr residues. We engineered and stably expressed chimeric receptors that consisted of the extracellular region of mouse CD6 and the cytoplasmic regions of either naturally occurring isoforms of human CD6, truncated proteins, or point mutants. We were able to demonstrate that of the nine Tyr residues in the cytoplasmic domain of the largest isoform CD6a, the two C-terminal Tyr residues (Tyr 629/662) are critical for the phosphorylation of CD6 following TCR cross-linking. Isoform CD6e, which is missing a region that contains two proline-rich motifs, is not phosphorylated. We further analyzed the ability of the different CD6 isoforms and truncated receptors to mobilize intracellular calcium after CD6/TCR co-ligation. All CD6 isoforms, including CD6e, as well as the truncation mutant Δ 555, which is missing approximately the C-terminal half of the cytoplasmic domain, are able to increase Ca²⁺ influx. Taken together, these results suggest that the region of CD6 which is critical for Ca²⁺ mobilization is located N-terminal from amino acid 555 and is therefore different from the region located at the C terminus of CD6, which is necessary for tyrosine phosphorylation.     

Insertion mutations of the RIIA Na+ channel reveal novel features of voltage gating and protein kinase A modulation

A linker insertion mutagenesis strategy was developed to probe functional subdomains of the RIIA Na⁺ channel α-subunit. We describe mutations within the first two repeat domains that provide new functional information for three segments of the channel structure.

1. The insertion of two alanine residues within the short peptide segment joining helices S4 and S5 in domain II had two effects: a depolarizing shift of steadystate activation and reduced single-channel currents. These results suggest that the peptide segment following the S4 voltage sensor is involved in the activation process and is facing the ion pore.
2. An insertion immediately N-terminal to the proposed transmembrane helix S1 in domain II shifted the steady-state activation in the depolarizing direction, suggesting a functional role in channel gating.
3. Insertions in the large, cytoplasmic loop between domains I and II affect two channel functions: inactivation and protein kinase A modulation. These results demonstrate that the linker insertion approach can provide novel insights into the structure-function relationships of large, multi-domain ion channel proteins.

     

Structure-function analysis of the TibA self-associating autotransporter reveals a modular organization

Some enterotoxigenic Escherichia coli strains express the TibA adhesin/invasin, a multifunctional autotransporter that mediates the autoaggregation of bacteria, biofilm formation, adhesion to cultured epithelial cells, and invasion of these cells. To elucidate the structure-function relationship in TibA, we generated mutants by transposon-based linker scanning mutagenesis and by site-directed mutagenesis. Several insertion mutants had a defect in either adhesion or autoaggregation. Mutants with a defect in autoaggregation were found in the N-terminal half of the extracellular domain, while mutants with a defect in adhesion were found in the C-terminal half. The deletion of the putative N-terminal autoaggregation domain abolished the autoaggregation of the bacteria but did not affect adhesion. The deletion of a proline-rich region located at the C terminus of the extracellular domain abolished the adhesion properties of TibA but did not affect invasion. This finding suggests that adhesion and invasion may rely on distinct mechanisms. Thus, our results reveal that TibA possesses a modular organization, with the extracellular domain being separated into an autoaggregation module and an adhesion module.     

Combined results from solution studies on intact influenza virus M1 protein and from a new crystal form of its N-terminal domain show that M1 is an elongated monomer

The amino-terminal domain of influenza A virus matrix protein (residues 1-164) was crystallized at pH 7 into a new crystal form in space group P1. This packing of the protein implies that M1(1-164) was monomeric in solution when it crystallized. Otherwise, the structure of the M1 fragment in the pH 7 crystals was the same as the monomers in crystals formed at pH 4 where crystal packing resulted in dimer formation [B. Sha and M. Luo, 1997, Nature Struct. Biol. 4, 239-244]. Analysis of intact M1 protein, the N-terminal domain, and the remaining C-terminal fragment (residues 165-252) in solution also showed that the N-terminal domain was monomeric with the same dimensions as determined from the crystal structure. Intact M1 protein was also monomeric but with an elongated shape due to the presence of the C-terminal part. Circular dichroism showed that the C-terminal part of M1 contained helical structure. A model for soluble M1 is presented, based on the assumption that the C-terminal domain is spherical, in which the N- and C-terminal domains are connected by a linker sequence which is available for proteolytic attack.     

Characterization of recombinant Bordetella pertussis adenylate cyclase toxins carrying passenger proteins.

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain (400 amino acid residues) into the cytosol of eukaryotic target cells, directly through the cytoplasmic membrane. We have previously shown that CyaA can be used as a vehicle to deliver CD8+ T-cell epitopes, inserted within the catalytic domain of the toxin, into antigen-presenting cells and can trigger specific class I-restricted cytotoxic T-cell (CTL) responses in vivo. To explore the tolerance of CyaA to insertion of polypeptides of larger size, we constructed and characterized different recombinant CyaA toxins with protein inserts of 87 to 206 amino acids in length. Several of these recombinant CyaA toxins were found to be invasive. Furthermore, we showed that the unfolding of the passenger protein is a prerequisite for the translocation of the recombinant toxins into eukaryotic cells. Our results highlight the remarkable tolerance of the CyaA toxin and suggest that CyaA might be used to deliver proteins into eukaryotic cells.