The cytoplasmic carboxy-terminal amino acid specifies cleavage of membrane TGF alpha into soluble growth factor.

Membrane-anchored transforming growth factor alpha (proTGF alpha) belongs to a group of transmembrane proteins whose extracellular domains are selectively cleaved and released into the medium. We demonstrate that the carboxy-terminal valine in the cytoplasmic tail of proTGF alpha is required for cleavage of the growth factor ectodomain in response to various activators. This cleavage process occurs outside Golgi or lysosomal locations, affects cell surface proTGF alpha, and requires little or no membrane traffic. We propose that cleavage and release of proTGF alpha ectodomain involve a specialized proteolytic system and depend on the recognition of a simple and specific determinant located in the proTGF alpha cytoplasmic tail.     

Sequence-independent targeting of transmembrane proteins synthesized within vaccinia virus factories to nascent viral membranes

The primary membrane of vaccinia virus, as well as those of other poxviruses, forms within a discrete cytoplasmic factory region. We recently determined the existence of an operative pathway from the endoplasmic reticulum within the virus factory to nascent viral membranes and demonstrated that a viral protein could be diverted from this pathway to Golgi membranes by the addition of COPII-binding sites (M. Husain, A. S. Weisberg, and B. Moss, Proc. Natl. Acad. Sci. USA, 103:19506-19511, 2006). Here we describe an investigation of the structural features that are required for transit of proteins to the viral membrane. Deletion of either the N-terminal domain or the C-terminal cytoplasmic tail from the conserved A9 protein did not prevent its incorporation into viral membranes, whereas deletion of the transmembrane domain resulted in its distribution throughout the cytoplasm. Nevertheless, replacement of the A9 transmembrane domain with the corresponding region of a nonpoxvirus transmembrane protein or of a vaccinia virus extracellular envelope protein allowed viral membrane targeting, indicating no requirement for a specific amino acid sequence. Remarkably, the epitope-tagged A9 transmembrane domain alone, as well as a heterologous transmembrane domain lacking a poxvirus sequence, was sufficient for viral membrane association. The data are consistent with a sequence-independent pathway in which transmembrane proteins that are synthesized within the virus factory and lack COPII or other binding sites that enable conventional endoplasmic reticulum exiting are incorporated into nascent viral membranes.     

A specific endoplasmic reticulum export signal drives transport of stem cell factor (Kitl) to the cell surface

Stem cell factor, also known as Kit ligand (Kitl), belongs to the family of dimeric transmembrane growth factors. Efficient cell surface presentation of Kitl is essential for the migration, proliferation, and survival of melanocytes, germ cells, hemopoietic stem cells, and mastocytes. Here we demonstrate that intracellular transport of Kitl to the cell surface is driven by a motif in the cytoplasmic tail that acts independently of the previously described basolateral sorting signal. Transport of Kitl to the cell surface is controlled at the level of the endoplasmic reticulum (ER) and requires a C-terminal valine residue positioned at a distance of 19-36 amino acids from the border between the transmembrane and cytoplasmic domains. Deletion or substitution of the valine with other hydrophobic amino acids results in ER accumulation and reduced cell surface transport of Kitl at physiological expression levels. When these mutant proteins are overexpressed in the ER, they are transported by bulk flow to the cell surface albeit at lower efficiency. A fusion construct between Kitl and the green fluorescent protein-labeled extracellular domain of a temperature-sensitive mutant of vesicular stomatitis virus G protein revealed the valine-dependent recruitment into coat protein complex II-coated ER exit sites and vesicular ER to Golgi transport in living cells. Thus the C-terminal valine defines a specific ER export signal in Kitl. It is responsible for the capture of Kitl at coat protein complex II-coated ER exit sites, leading to subsequent cell surface transport under physiological conditions.     

Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

Dysferlin and calpain are important mediators of the emergency response to repair plasma membrane injury. Our previous research revealed that membrane injury induces cleavage of dysferlin to release a synaptotagmin-like C-terminal module we termed mini-dysferlin_C72. Here we show that injury-activated cleavage of dysferlin is mediated by the ubiquitous calpains via a cleavage motif encoded by alternately spliced exon 40a. An exon 40a–specific antibody recognizing cleaved mini-dysferlin_C72 intensely labels the circumference of injury sites, supporting a key role for dysferlin_Exon40a isoforms in membrane repair and consistent with our evidence suggesting that the calpain-cleaved C-terminal module is the form specifically recruited to injury sites. Calpain cleavage of dysferlin is a ubiquitous response to membrane injury in multiple cell lineages and occurs independently of the membrane repair protein MG53. Our study links calpain and dysferlin in the calcium-activated vesicle fusion of membrane repair, placing calpains as upstream mediators of a membrane repair cascade that elicits cleaved dysferlin as an effector. Of importance, we reveal that myoferlin and otoferlin are also cleaved enzymatically to release similar C-terminal modules, bearing two C2 domains and a transmembrane domain. Evolutionary preservation of this feature highlights its functional importance and suggests that this highly conserved C-terminal region of ferlins represents a functionally specialized vesicle fusion module.     

Structure and orientation of halorhodopsin in the membrane: a proteolytic fragmentation study.

Halorhodopsin (HR), the light-driven chloride pump in halobacteria, was digested with various proteolytic enzymes. As expected, carboxypeptidase A removed 14 amino acids from the C-terminal tail of detergent-solubilized HR, producing a fragment of 25.2 kd in size. Membrane-associated HR could be digested as well, but not in right-side-out sealed cell envelope vesicles. We conclude, therefore, that the orientation of HR in the cytoplasmic membrane is such that the C-terminal tail faces the cytoplasmic side. Tryptic digestion of detergent-solubilized HR resulted in the removal of the same C-terminal segment, but also in the production of two more cleavage products (molecular masses of 20.9 and 16.8 kd respectively). These cleavage sites were determined by amino acid sequencing of the newly produced N termini, and they turned out to be within interhelical loops in an earlier proposed structural model for HR. Incubation with chymotrypsin and thermolysin yielded different sites of cleavage, but also in regions which were proposed to be accessible on the surface of the protein. Since the results show that three of six proposed interhelical loop segments contain proteolytic digestion sites, they support the proposed structural model for HR.     

Cell-associated episialin is a complex containing two proteins derived from a common precursor.

cDNA for the epithelial sialomucin episialin encodes a transmembrane molecule with a large extracellular domain, which mainly consists of repeats of 20 amino acids. Here we confirm the existence of a previously proposed proteolytic cleavage of episialin that occurs in the endoplasmic reticulum (Hilkens, J., and Buijs, F. (1988) J. Biol. Chem. 263, 4215-4222) and show that a similar cleavage takes place in in vitro translation systems. Using in vitro translation of truncated mRNAs, we map the cleavage site to a region located between 71 and 53 amino acids upstream of the transmembrane domain. Analysis of a mutant, in which this region has been deleted, indicates that the cleavage sites used in vitro and in vivo are identical or in close proximity. Both cleavage products remain associated although they are not linked through disulfide bonds. Therefore, the subunit derived from the N terminus, which represents the actual mucin-like domain, remains indirectly anchored to the cell membrane as a result of its interaction with the C-terminal subunit.     

Role of a small cytoplasmic domain in the establishment of serine chemoreceptor membrane topology.

The Escherichia coli serine chemoreceptor takes on a simple membrane topology with two transmembrane segments separating cytoplasmically disposed N and C termini from a central periplasmic domain. We investigated the role of the small N-terminal cytoplasmic domain in membrane insertion using alkaline phosphatase gene fusions. Mutations eliminating the positive charge of the domain altered insertion dramatically, with reciprocal effects on hybrids with periplasmic and C-terminal cytoplasmic fusion junctions. Efficient export of the normally cytoplasmic C-terminal domain required that, in addition to the N-terminal changes, a short amphiphatic sequence at the beginning of the C-terminal domain be also absent. These findings document the importance of the positive character of the N-terminal domain in chemoreceptor membrane insertion and imply that partially redundant sequence information controls the orientation of the second transmembrane segment.     

Targeting of a lysosomal membrane protein: a tyrosine-containing endocytosis signal in the cytoplasmic tail of lysosomal acid phosphatase is necessary and sufficient for targeting to lysosomes.

Lysosomal acid phosphatase (LAP) is synthesized as a transmembrane protein with a short carboxy-terminal cytoplasmic tail of 19 amino acids, and processed to a soluble protein after transport to lysosomes. Deletion of the membrane spanning domain and the cytoplasmic tail converts LAP to a secretory protein, while deletion of the cytoplasmic tail as well as substitution of tyrosine 413 within the cytoplasmic tail against phenylalanine causes accumulation at the cell surface. A chimeric polypeptide, in which the cytoplasmic tail of LAP was fused to the ectoplasmic and transmembrane domain of hemagglutinin is rapidly internalized and tyrosine 413 of the LAP tail is essential for internalization of the fusion protein. A chimeric polypeptide, in which the membrane spanning domain and cytoplasmic tail of LAP are fused to the ectoplasmic domain of the Mr 46 kd mannose 6-phosphate receptor, is rapidly transported to lysosomes, whereas wild type receptor is not transported to lysosomes. We conclude that a tyrosine containing endocytosis signal in the cytoplasmic tail of LAP is necessary and sufficient for targeting to lysosomes.     

Structure and expression of the membrane proteoglycan betaglycan, a component of the TGF-beta receptor system.

We describe the primary structure of rat betaglycan, a polymorphic membrane-anchored proteoglycan with high affinity for transforming growth factor-beta (TGF-beta). As deduced from its cDNA sequence, the 853 amino acid core protein of betaglycan has an extracellular domain with clustered sites for potential attachment of glycosaminoglycan chains. These chains are dispensable for TGF-beta binding to the core protein. The transmembrane region and the short cytoplasmic tail of betaglycan are very similar to these regions in human endoglin, an endothelial cell membrane glycoprotein involved in intercellular recognition. The ectodomain of betaglycan can be released as a soluble proteoglycan; a potential cleavage site near the transmembrane region is identical to the highly regulated cleavage site of the membrane-anchored transforming growth factor-alpha precursor. The unique features of betaglycan suggest important roles in cell interaction with TGF-beta.     

Membrane topology and quaternary structure of cardiac gap junction ion channels.

The membrane topology and quaternary structure of rat cardiac gap junction ion channels containing alpha 1 connexin (i.e. Cx43) have been examined using anti-peptide antibodies directed to seven different sites in the protein sequence, cleavage by an endogenous protease in heart tissue and electron microscopic image analysis of native and protease-cleaved two-dimensional membrane crystals of isolated cardiac gap junctions. Specificity of the peptide antibodies was established using dot immunoblotting, Western immunoblotting, immunofluorescence and immunoelectron microscopy. Based on the folding predicted by hydropathy analysis, five antibodies were directed to sites in cytoplasmic domains and two antibodies were directed to the two extracellular loop domains. Isolated gap junctions could not be labeled by the two extracellular loop antibodies using thin-section immunogold electron microscopy. This is consistent with the known narrowness of the extracellular gap region that presumably precludes penetration of antibody probes. However, cryo-sectioning rendered the extracellular domains accessible for immunolabeling. A cytoplasmic "loop" domain of at least Mr = 5100 (residues (101 to 142) is readily accessible to peptide antibody labeling. The native Mr = 43,000 protein can be protease-cleaved on the cytoplasmic side of the membrane, resulting in an Mr approximately 30,000 membrane-bound fragment. Western immunoblots showed that protease cleavage occurs at the carboxy tail of the protein, and the cleavage site resides between amino acid residues 252-271. Immunoelectron microscopy demonstrated that the Mr approximately 13,000 carboxy-terminal peptide(s) is released after protease cleavage and does not remain attached to the Mr approximately 30,000 membrane-bound fragment via non-covalent interactions. Electron microscopic image analysis of two-dimensional membrane crystals of cardiac gap junctions revealed that the ion channels are formed by a hexagonal arrangement of protein subunits. This quaternary arrangement is not detectably altered by protease cleavage of the alpha 1 polypeptide. Therefore, the Mr approximately 13,000 carboxyterminal domain is not involved in forming the transmembrane ion channel. The similar hexameric architecture of cardiac and liver gap junction connexins indicates conservation in the molecular design of the gap junction channels formed by alpha or beta connexins.     

Mutations at the P1' position of Notch1 decrease intracellular domain stability rather than cleavage by gamma-secretase

Gamma-secretase is a unique protease which cleaves within the transmembrane domain of several substrate proteins. Among gamma-secretase substrates are members of the Notch family of receptors and the amyloid precursor protein. In this study we used a cell-free Notch-cleavage assay and specific gamma-secretase inhibitors to study the cleavage of Notch by gamma-secretase. Using this assay, we found that, in contrast to previous reports, the presence of valine at the P1(') position of Notch1 is not required for gamma-secretase cleavage. Our results suggest that the presence of valine at the N-terminus of the Notch intracellular domain cleavage product is important for its stability. Thus it appears that Notch cleavage is very similar to APP cleavage with respect to the lack of sequence specificity.     

Modulation of cell surface transport and lipid raft localization by the cytoplasmic tail of the influenza virus hemagglutinin

Viral glycoproteins are highly variable in their primary structure, but on the other hand feature a high functional conservation to fulfil their versatile tasks during the pathogenic life cycle. Typically, all protein domains are optimized in that indispensable functions can be assigned to small conserved motifs or even individual amino acids. The cytoplasmic tail of many viral spike proteins, although of particular relevance for the virus biology, is often only insufficiently characterized. Hemagglutinin (HA), the receptor‐binding protein of the influenza virus comprises a short cytoplasmic tail of 13 amino acids that exhibits three highly conserved palmitoylation sites. However, the particular importance of these modifications and the tail in general for intracellular trafficking and lateral membrane organization remains elusive. In this study, we generated HA core proteins consisting of transmembrane domain, cytoplasmic tail and a minor part of the ectodomain, tagged with a yellow fluorescent protein. Different mutation and truncation variants of these chimeric proteins were investigated using confocal microscopy, to characterize the role of cytoplasmic tail and palmitoylation for the intracellular trafficking to plasma membrane and Golgi apparatus. In addition, we assessed raft partitioning of the variants by Foerster resonance energy transfer with an established raft marker. We revealed a substantial influence of the cytoplasmic tail length on the intracellular distribution and surface exposure of the proteins. A complete removal of the tail hampers a physiological trafficking of the protein, whereas a partial truncation can be compensated by cytoplasmic palmitoylations. Plasma membrane raft partitioning on the other hand was found to imperatively require palmitoylations, and the cysteine at position 551 turned out to be of most relevance. Our data shed further light on the tight interconnection between cytoplasmic elements and intracellular trafficking and suggest a function of HA palmitoylations in both lateral sorting and anterograde trafficking of the glycoprotein.     

Important role for the transmembrane domain of severe acute respiratory syndrome coronavirus spike protein during entry

The spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for receptor binding and membrane fusion. It contains a highly conserved transmembrane domain that consists of three parts: an N-terminal tryptophan-rich domain, a central domain, and a cysteine-rich C-terminal domain. The cytoplasmic tail of S has previously been shown to be required for assembly. Here, the roles of the transmembrane and cytoplasmic domains of S in the infectivity and membrane fusion activity of SARS-CoV have been studied. SARS-CoV S-pseudotyped retrovirus (SARSpp) was used to measure S-mediated infectivity. In addition, the cell-cell fusion activity of S was monitored by a Renilla luciferase-based cell-cell fusion assay. S(VSV-Cyt), an S chimera with a cytoplasmic tail derived from vesicular stomatitis virus G protein (VSV-G), and S(MHV-TMDCyt), an S chimera with the cytoplasmic and transmembrane domains of mouse hepatitis virus, displayed wild-type-like activity in both assays. S(VSV-TMDCyt), a chimera with the cytoplasmic and transmembrane domains of VSV-G, was impaired in the SARSpp and cell-cell fusion assays, showing 3 to 25% activity compared to the wild type, depending on the assay and the cells used. Examination of the oligomeric state of the chimeric S proteins in SARSpp revealed that S(VSV-TMDCyt) trimers were less stable than wild-type S trimers, possibly explaining the lowered fusogenicity and infectivity.     

The distal cytoplasmic tail of the influenza A M2 protein dynamically extends from the membrane

The influenza A M2 protein is a multifunctional membrane-associated homotetramer that orchestrates several essential events in the viral infection cycle. The monomeric subunits of the M2 homotetramer consist of an N-terminal ectodomain, a transmembrane domain, and a C-terminal cytoplasmic domain. The transmembrane domain forms a four-helix proton channel that promotes uncoating of virions upon host cell entry. The membrane-proximal region of the C-terminal domain forms a surface-associated amphipathic helix necessary for viral budding. The structure of the remaining ~34 residues of the distal cytoplasmic tail has yet to be fully characterized despite the functional significance of this region for influenza infectivity. Here, we extend structural and dynamic studies of the poorly characterized M2 cytoplasmic tail. We used SDSL-EPR to collect site-specific information on the mobility, solvent accessibility, and conformational properties of residues 61–70 of the full-length, cell-expressed M2 protein reconstituted into liposomes. Our analysis is consistent with the predominant population of the C-terminal tail dynamically extending away from the membranes surface into the aqueous medium. These findings provide insight into the hypothesis that the C-terminal domain serves as a sensor that regulates how M2 protein participates in critical events in the viral infection cycle.     

Molecular signals for phosphatidylinositol modification of the Qa-2 antigen

Most cell surface proteins are anchored to the cell bilayer by hydrophobic membrane-spanning domains. Recently it has been shown that a small class of molecules are attached to cell surfaces via a phosphatidylinositol moiety covalently linked to the C-terminus of the mature processed polypeptide. The molecular signals that identify a polypeptide for phosphatidylinositol (PI) attachment have not been well defined in any system, but are thought to reside in the C-terminus of the primary translation product. We report that all the signals responsible for PI anchoring of Qa-2 Ag are confined to the 36 C-terminal residues of the precursor proteins. To investigate further the features that signal cleavage and PI addition, we have studied mutants of two closely related murine class I MHC molecules: the PI-linked Ag, Q9b, from the Qa-2 Ag family, and the integral membrane transplantation antigen, H-2Ld. The addition of 15 amino acids to the three residue long cytoplasmic domain of Q9b or the mutation of Asp295 found in its C-terminal hydrophobic domain to Val converts this molecule into an integral membrane protein. However, the introduction of a short three residue cytoplasmic tail and Asp295 into the transmembrane domain of H-2Ld does not convert this molecule to a PI-linked one. The results of these analyses suggest that the PI-processing signals may depend on overall conformation, hydrophobicity, and length of the C-terminal domain of the precursor protein. In addition these data indicate that PI anchoring of class I Ag requires more than two mutational steps and may have been selected during the evolution.     

Identification and characterization of splice variants of the human P2X7 ATP channel

The P2X7 channel is a member of the P2X family of ligand-gated ion channels which respond to ATP as the endogenous agonist. Studies suggest that P2X7 has a potentially pivotal role in inflammatory responses largely stemming from its role in mediating the release of IL-1beta in response to ATP. We report the identification of seven variants of human P2X7 which result from alternative splicing. Two of these variants (one lacking the first transmembrane domain, the second lacking the entire cytoplasmic tail) were compared to the full-length channel. Real-time PCR analysis demonstrated that both variants were expressed in various tissues and that the cytoplasmic tail deleted variant is highly expressed. Deletion of the first transmembrane domain resulted in a non-functional channel. Deletion of the cytoplasmic tail did not affect ion movement but severely affected the ability to form a large pore and to induce activation of caspases.     

A tyrosine motif in the cytoplasmic domain of mason-pfizer monkey virus is essential for the incorporation of glycoprotein into virions

Mason-Pfizer monkey virus (M-PMV) encodes a transmembrane (TM) glycoprotein with a 38-amino-acid-long cytoplasmic domain. After the release of the immature virus, a viral protease-mediated cleavage occurs within the cytoplasmic domain, resulting in the loss of 17 amino acids from the carboxy terminus. This maturational cleavage occurs between a histidine at position 21 and a tyrosine at position 22 in the cytoplasmic domain of the TM protein. We have demonstrated previously that a truncated TM glycoprotein with a 21-amino-acid-long cytoplasmic tail showed enhanced fusogenicity but could not be incorporated into virions. These results suggest that postassembly cleavage of the cytoplasmic domain removes a necessary incorporation signal and activates fusion activity. To investigate the contribution of tyrosine residues to the function of the glycoprotein complex and virus replication, we have introduced amino acid substitutions into two tyrosine residues found in the cytoplasmic domain. The effects of these mutations on glycoprotein biosynthesis and function, as well as on virus infectivity, have been examined. Mutation of tyrosine 34 to alanine had little effect on glycoprotein function. In contrast, substitutions at tyrosine 22 modulated fusion activity in either a positive or negative manner, depending on the substituting amino acid. Moreover, any nonaromatic substitution at this position blocked glycoprotein incorporation into virions and abolished infectivity. These results demonstrate that M-PMV employs a tyrosine signal for the selective incorporation of glycoprotein into budding virions. Antibody uptake studies show that tyrosine 22 is part of an efficient internalization signal in the cytoplasmic domain of the M-PMV glycoprotein that can also be positively and negatively influenced by changes at this site.     

Flexibility of the cytoplasmic domain of the anion exchange protein, band 3, in human erythrocytes.

The rotational flexibility of the cytoplasmic domain of band 3, in the region that is proximal to the inner membrane surface, has been investigated using a combination of time-resolved optical anisotropy (TOA) and saturation-transfer electron paramagnetic resonance (ST-EPR) spectroscopies. TOA studies of rotational diffusion of the transmembrane domain of band 3 show a dramatic decrease in residual anisotropy following cleavage of the link with the cytoplasmic domain by trypsin (E. A. Nigg and R. J. Cherry, 1980, Proc. Natl. Acad. Sci. U.S.A. 77:4702-4706). This result is compatible with two independent hypotheses: 1) trypsin cleavage leads to dissociation of large clusters of band 3 that are immobile on the millisecond time scale, or 2) trypsin cleavage leads to release of a constraint to uniaxial rotational diffusion of the transmembrane domain. ST-EPR studies at X- and Q-band microwave frequencies detect rotational diffusion of the transmembrane domain of band 3 about the membrane normal axis of reasonably large amplitude that does not change upon cleavage with trypsin. These ST-EPR results are not consistent with dissociation of clusters of band 3 as a result of cleavage with trypsin. Global analyses of the ST-EPR data using a newly developed algorithm indicate that any constraint to rotational diffusion of the transmembrane domain of band 3 via interactions of the cytoplasmic domain with the membrane skeleton must be sufficiently weak to allow rotational excursions in excess of 32 degrees full-width for a square-well potential. In support of this result, analyses of the TOA data in terms of restricted amplitude uniaxial rotational diffusion models suggest that the membrane-spanning domain of that population of band 3 that is linked to the membrane skeleton is constrained to diffuse in a square-well of approximately 73 degrees full-width. This degree of flexibility may be necessary for providing the unique mechanical properties of the erythrocyte membrane.