The transmembrane segment of ryanodine receptor contains an intracellular membrane retention signal for Ca(2+) release channel.

The ryanodine receptor (RyR) is a large homotetrameric protein with a hydrophobic domain at the C-terminal end that resides in the endoplasmic reticulum (ER) or sarcoplasmic reticulum membrane and forms the conduction pore of a Ca(2+) release channel. Our previous studies showed that RyR expressed in heterologous cells localized to the ER membrane. Confocal microscopic imaging indicated that the ER retention signal is likely present within the C-terminal portion of RyR, a region that contains four putative transmembrane segments. To identify the amino acid sequence responsible for ER retention of RyR, we expressed fusion proteins containing intercellular adhesion molecule (ICAM), various fragments of RyR, and green fluorescent protein (GFP) in Chinese hamster ovary and COS-7 cells. ICAM is a plasma membrane-resident glycoprotein and serves as a reporter for protein trafficking to the cell surface membrane. Imaging analyses indicated that ICAM-GFP fusion proteins with RyR sequence preceding the four transmembrane segments, ICAM-RyR-(3661-3993)-GFP, and with RyR sequence corresponding to transmembrane segments 1, 2, and 3, ICAM-RyR-(4558-4671)-GFP and ICAM-RyR-(4830-4919)-GFP, were localized to the plasma membrane; fusion proteins containing the fourth transmembrane segment of RyR, ICAM-RyR-(4913-4943)-GFP, were retained in the ER. Biochemical assay showed that ICAM-RyR-GFP fusion proteins that target to the plasma membrane are fully glycosylated, and those retained in the intracellular membrane are core-glycosylated. Together our data indicate that amino acids 4918-4943 of RyR contain the signal sequence for ER retention of the Ca(2+) release channel.     

Colocalized transmembrane determinants for ER degradation and subunit assembly explain the intracellular fate of TCR chains

The intracellular fate of T cell antigen receptor (TCR) subunits (alpha beta gamma delta epsilon zeta 2) is determined by their assembly in the endoplasmic reticulum (ER). To study the structural bases for this tight correlation between assembly and intracellular fate, we sought to define the nature of determinants for both ER degradation and subunit assembly within the TCR-alpha chain. We found that a 9 amino acid transmembrane sequence of the TCR-alpha chain, containing 2 critical charged residues, was sufficient to cause ER degradation when placed in the context of the Tac antigen, used here as a reporter protein. CD3-delta assembled with chimeric proteins containing this short transmembrane sequence, and this assembly resulted in abrogation of targeting for ER degradation. Thus, the colocalization of determinants for ER degradation and sites of subunit interactions explains how the fate of some newly synthesized TCR chains can be decided on the basis of their assembly status.     

Molecular cloning and sequence analysis of human genomic DNA encoding a novel membrane protein which exhibits a slowly activating potassium channel activity

The amino acid sequence for a novel human membrane protein that induces selective potassium permeation by membrane depolarization was deduced by molecular cloning and sequence analysis of its genomic DNA. This protein consists of 129 amino acid residues and shares several structural characteristics with the rat counterpart. These include a single putative transmembrane domain surrounded by many charged amino acid residues, two potential N-glycosylation sites at the amino-terminal portion and a single cysteine residue at the carboxyl-terminal portion. The transmembrane domain and its flanking carboxyl-terminal sequence are highly conserved between the human and rat sequences. Because the slowly activating potassium current elicited by the human protein on its expression in Xenopus oocytes is indistinguishable from that induced by the rat protein, the sequence conserved at the transmembrane domain and its following sequence should play an essential role in the induction of selective K+ permeation.     

Short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum

The adenoviral transmembrane E3/19K glycoprotein is a resident of the endoplasmic reticulum. Here we show that the last six amino acid residues of the 15-membered cytoplasmic tail are necessary and sufficient for the ER retention. These residues can be transplanted onto the cytoplasmic tail of other membrane-bound proteins such that ER residency is conferred. Deletion analysis demonstrated that no single amino acid residue is responsible for the retention. The identified structural motif must occupy the extreme COOH-terminal position to be functional. An endogenous transmembrane ER protein, UDP-glucuronosyltransferase, also contains a retention signal in its cytoplasmic tail. We suggest that short linear sequences occupying the extreme COOH-terminal position of transmembrane ER proteins serve as retention signals.     

Retention of subunits of the oligosaccharyltransferase complex in the endoplasmic reticulum

Membrane proteins of the endoplasmic reticulum (ER) may be localized to this organelle by mechanisms that involve retention, retrieval, or a combination of both. For luminal ER proteins, which contain a KDEL domain, and for type I transmembrane proteins carrying a dilysine motif, specific retrieval mechanisms have been identified. However, most ER membrane proteins do not contain easily identifiable retrieval motifs. ER localization information has been found in cytoplasmic, transmembrane, or luminal domains. In this study, we have identified ER localization domains within the three type I transmembrane proteins, ribophorin I (RI), ribophorin II (RII), and OST48. Together with DAD1, these membrane proteins form an oligomeric complex that has oligosaccharyltransferase (OST) activity. We have previously shown that ER retention information is independently contained within the transmembrane and the cytoplasmic domain of RII, and in the case of RI, a truncated form consisting of the luminal domain was retained in the ER. To determine whether other domains of RI carry additional retention information, we have generated chimeras by exchanging individual domains of the Tac antigen with the corresponding ones of RI. We demonstrate here that only the luminal domain of RI contains ER retention information. We also show that the dilysine motif in OST48 functions as an ER localization motif because OST48 in which the two lysine residues are replaced by serine (OST48ss) is no longer retained in the ER and is found instead also at the plasma membrane. OST48ss is, however, retained in the ER when coexpressed with RI, RII, or chimeras, which by themselves do not exit from the ER, indicating that they may form partial oligomeric complexes by interacting with the luminal domain of OST48. In the case of the Tac chimera containing only the luminal domain of RII, which by itself exits from the ER and is rapidly degraded, it is retained in the ER and becomes stabilized when coexpressed with OST48.     

Membrane protein retention in the yeast Golgi apparatus: dipeptidyl aminopeptidase A is retained by a cytoplasmic signal containing aromatic residues

The mechanism by which yeast dipeptidyl aminopeptidase (DPAP) A, type II integral membrane protein, is retained in the late Golgi apparatus has been investigated. Prior work demonstrated that the 118-amino acid cytoplasmic domain is both necessary and sufficient for Golgi retention and that mutant or overexpressed DPAP A no longer retained in the Golgi was delivered directly to the vacuolar membrane (Roberts, C. J., S. F. Nothwehr, and T. H. Stevens. 1992. J. Cell Biol. 119:69-83). Replacement of the DPAP A transmembrane domain with a synthetic hydrophobic sequence did not affect either Golgi retention of DPAP A or vacuolar delivery of the retention-defective form of DPAP A. These results indicate that the DPAP A transmembrane domain is not involved in either Golgi retention or targeting of this membrane protein. A detailed mutational analysis of the cytoplasmic domain of DPAP A indicated that the most important elements for retention were within the eight residue stretch 85-92. A 10-amino acid region from DPAP A (81- 90) was sufficient for Golgi retention of alkaline phosphatase, a type II vacuolar membrane protein. Detailed mutational analysis within this 10-amino acid sufficient region demonstrated that a Phe-X-Phe-X-Asp motif was absolutely required for efficient retention. The efficiency of Golgi retention via the DPAP A signal could be diminished by overexpression of wild type but not retention-defective versions of Kex2p, another late Golgi membrane protein, suggesting that multiple Golgi membrane proteins may be retained by a common machinery. These results imply a role for a cytoplasmic signal involving aromatic residues in retention of late Golgi membrane proteins in the yeast Saccharomyces cerevisiae.     

Characterization and purification of the 94-kDa glucose-regulated protein

Increased synthesis of so-called glucose-regulated proteins (grp) of 78 and 94 kDa occurs in mammalian cells exposed to a variety of agents, including 2-mercaptoethanol, tunicamycin, agents which perturb calcium homeostasis, and amino acid analogs. Herein we describe a number of properties of 94-kDa grp (grp 94) and present a method for its purification to homogeneity. The protein, within the endoplasmic reticulum (ER), is modified by the addition of high mannose-containing oligosaccharides. The predicted amino acid sequence of grp 94, as determined by others, has revealed the protein to contain a putative transmembrane domain near its amino terminus, but in addition, a potential endoplasmic reticulum retention sequence (KDEL) at its COOH terminus. Consequently, the question of whether grp 94 exists as a transmembrane or luminal protein of the ER remains controversial. Results using isolated microsomes subjected to either limited proteolysis or lactoperoxidase-mediated iodination were consistent with the idea that the grp is a transmembrane protein. On the other hand, using the method of sodium carbonate extraction, grp 94 exhibited properties of both a luminal and integral membrane protein. These results raise the question of whether there exist two different forms of grp 94 within the ER.     

Localization of ribophorin II to the endoplasmic reticulum involves both its transmembrane and cytoplasmic domains

Proteins that are concentrated in specific compartments of the endomembrane system in order to exert their organelle-specific function must possess specific localization signals that prevent their transport to distal regions of the exocytic pathway. Some resident proteins of the endoplasmic reticulum (ER) that are known to escape with low efficiency from this organelle to a post ER compartment are recognized by a recycling receptor and brought back to their site of residence. Other ER proteins, however, appear to be retained in the ER by mechanisms that operate in the organelle itself. The mammalian oligosaccharyltransferase (OST) is a protein complex that effects the cotranslational N-glycosylation of newly synthesized polypeptides, and is composed of at least four rough ER-specific membrane proteins: ribophorins I and II (RI and RII), OST48, and Dadl. The mechanism(s) by which the subunits of this complex are retained in the ER are not well understood. In an effort to identify the domains within RII responsible for its ER localization we have studied the fate of chimeric proteins in which one or more RII domains were replaced by the corresponding ones of the Tac antigen, the latter being a well characterized plasma membrane protein that lacks intrinsic ER retention signals and serves to provide a neutral framework for the identification of retention signals in other proteins. We found that the luminal domain of RII by itself does not contain retention information, while the cytoplasmic and transmembrane domains contain independent ER localization signals. We also show that the retention function of the transmembrane domain is strengthened by the presence of a flanking luminal region consisting of 15 amino acids.     

Negatively charged residues in the IgM stop-transfer effector sequence regulate transmembrane polypeptide integration

A non-hydrophobic sequence that contributes to the biogenesis of a transmembrane protein is termed a stop-transfer effector (STE). To examine the mechanism of STE-mediated stop-transfer, a series of fusion proteins were constructed containing variants of a putative STE from murine IgM fused to an otherwise translocated hydrophobic sequence. Unexpectedly, the fraction of molecules adopting transmembrane topology was insensitive to many amino acid substitutions within the STE sequence but varied directly with the number of negative charges. Furthermore, when present at the amino terminus of a reporter, mutants were observed that adopted type I (amino terminus lumenal) and type II (amino terminus cytoplasmic) transmembrane topologies, demonstrating that the STE sequence can be located at either side of the endoplasmic reticulum membrane. Our results suggest that recognition of a broad structural feature formed primarily by negatively charged residues within the STE halts translocation and triggers membrane integration, even when the negative charges end up on the cytoplasmic side of the membrane. Since functional STE sequences photocross-link to two membrane proteins not previously identified at the translocon, these unique proteins are presumably involved in recognizing STE sequences and/or facilitating STE function.     

The transmembrane domain of the molecular chaperone Cosmc directs its localization to the endoplasmic reticulum

The molecular basis for retention of integral membrane proteins in the endoplasmic reticulum (ER) is not well understood. We recently discovered a novel ER molecular chaperone termed Cosmc, which is essential for folding and normal activity of the Golgi enzyme T-synthase. Cosmc, a type II single-pass transmembrane protein, lacks any known ER retrieval/retention motifs. To explore specific ER localization determinants in Cosmc we generated a series of Cosmc mutants along with chimeras of Cosmc with a non-ER resident type II protein, the human transferrin receptor. Here we show that the 18 amino acid transmembrane domain (TMD) of Cosmc is essential for ER localization and confers ER retention to select chimeras. Moreover, mutations of a single Cys residue within the TMD of Cosmc prevent formation of disulfide-bonded dimers of Cosmc and eliminate ER retention. These studies reveal that Cosmc has a unique ER-retention motif within its TMD and provide new insights into the molecular mechanisms by which TMDs of resident ER proteins contribute to ER localization.     

Generalized transposon shuttle mutagenesis in Neisseria gonorrhoeae: a method for isolating epithelial cell invasion-defective mutants

Hybrid genes were constructed to express bifunctional hybrid proteins in which staphyloccal nuclease A with or without an amino-terminai OmpA signal sequence was fused with TEM β-lactamase (at the carboxyl terminal side) using the signal peptide of the major outer membrane lipoprotein of Escherichia coli as an internal linker. The hybrid proteins were found to be inserted in the membrane. Orientation of the hybrid protein with the OmpA signal peptide showed that the nuclease was translocated into the periplasm and the β-lactamase remained in the cytoplasm. This indicates that the cleavable OmpA signal peptide served as a secretory signal for nuclease and the internal lipoprotein signal served as the transmembrane anchor, in the absence of the OmpA signal sequence the topology of the hybrid protein was reversed indicating that the internal lipoprotein signal peptide initially served as the signal peptide for the secretion of the carboxy terminal β-lactamase domain across the membrane and subsequently as a membrane anchoring signal. The role of charged amino acids in the translocation and transmembrane orientation of membrane proteins was also analysed by introducing charged amino acids to either or both sides of the internal lipoprotein signal sequence in the bifunctional hybrid proteins in the absence of the amino-terminal signal sequence. Introduction of two lysine residues at the carboxy-terminal side of the internal signal sequence reversed the topology of the transmembrane protein by translocating the aminoterminal nuclease domain across the membrane, leaving the carboxyl terminal β-actamase domain in the cytoplasm. When three more lysine residues were added to the amino-terminal side of the internal signal sequence of the same construct the membrane topology flipped back to the original orientation. A similar reversion of the topology could be obtained by introducing negatively charged residues at the amino-terminal side of the internal signal sequence. Present results demonstrate for the first time that a bifunctional transmembrane protein can be engineered to assume either of the two opposite orientations and that charge balance around the transmembrane domain is a major factor in controlling the topology of a transmembrane protein.     

Complete amino acid sequence of PO protein in bovine peripheral nerve myelin

The P0 protein is a major structural glycoprotein of molecular weight 28,000 in peripheral nerve myelin. The complete amino acid sequence of bovine P0 protein was determined. The polypeptide chain consists of 219 amino acid residues and includes a highly hydrophobic domain (residues 125-150) in the middle, which probably represents a transmembrane segment. The amino terminal domain (residues 1-124) is relatively hydrophobic, but contains a negatively charged carbohydrate chain at Asn93. This domain is most likely located on the extracellular side of the membrane and may contribute to formation of the myelin intraperiod line by hydrophobic and electrostatic interactions. On the other hand, the basic carboxyl-terminal domain (residues 151-219) may protrude from the cytoplasmic side of the membrane and is probably involved together with basic proteins in the formation of the major myelin dense line through electrostatic interaction with acidic lipids in the membrane. The few interspecies amino acid variations between the bovine P0 and the rat P0 sequences, deduced from the cDNA (Lemke, G., and Axel, R. (1985) Cell 40, 501-508), indicate that the P0 protein is conserved across species.     

The amino-terminal structures that determine topological orientation of cytochrome P-450 in microsomal membrane.

We previously showed that the amino-terminal region of P-450 is responsible not only for targeting to endoplasmic reticulum (ER) membrane but also for stable anchoring to the membrane. In the present study, we introduced several mutations or deletions into the signal-anchor region of the chimeric proteins in which the amino-terminal regions of two forms of cytochrome P-450 were fused to the mature portion of interleukin 2. The amino-terminal acidic amino acid residues were replaced with basic amino acid residues or the hydrophobic core sequences were partially deleted, and these mutant proteins were assayed in vitro for their capacity to be inserted into or translocated across the ER membrane. The proteins that received the former manipulations were processed and the IL-2 portion was translocated across the membrane. In one case, the processing did not occur, thereby enabling the chimeric protein to anchor on the luminal side of the ER. Those that received the latter manipulation were also processed and the IL-2 portion translocated across the ER. These results strongly suggest that the signal-anchor function is determined both by the amino-terminal charged amino acid residues and by the length of the hydrophobic stretch.     

Positive charges in the cytoplasmic domain of Escherichia coli leader peptidase prevent an apolar domain from functioning as a signal.

Leader peptidase, an integral transmembrane protein of Escherichia coli, requires two apolar topogenic elements for its membrane assembly: a 'hydrophobic helper' and an internal signal. The highly basic cytoplasmic region between these domains is a translocation poison sequence, which we have shown blocks the function of a preceding signal sequence. We have used oligonucleotide-directed mutagenesis to remove positively charged residues within this polar domain to determine if it is the basic character in this region that has the negative effect on translocation. Our results show that mutations that remove two or more of the positively charged residues within the polar region no longer block membrane assembly of leader peptidase. In addition, when the translocation poison domain (residues 30-52) is replaced with six lysine residues, the preceding apolar domain cannot function as an export signal, whereas it can with six glutamic acids. Thus, positively charged residues within membrane proteins may have a major role in determining the function of hydrophobic domains in membrane assembly.     

Biogenesis and transmembrane topology of the CHIP28 water channel at the endoplasmic reticulum

CHIP28 is a 28-kD hydrophobic integral membrane protein that functions as a water channel in erythrocytes and renal tubule epithelial cell membranes. We examined the transmembrane topology of CHIP28 in the ER by engineering a reporter of translocation (derived from bovine prolactin) into nine sequential sites in the CHIP28 coding region. The resulting chimeras were expressed in Xenopus oocytes, and the topology of the reporter with respect to the ER membrane was determined by protease sensitivity. We found that although hydropathy analysis predicted up to seven potential transmembrane regions, CHIP28 spanned the membrane only four times. Two putative transmembrane helices, residues 52-68 and 143-157, reside on the lumenal and cytosolic surfaces of the ER membrane, respectively. Topology derived from these chimeric proteins was supported by cell-free translation of five truncated CHIP28 cDNAs, by N-linked glycosylation at an engineered consensus site in native CHIP28 (residue His69), and by epitope tagging of the CHIP28 amino terminus. Defined protein chimeras were used to identify internal sequences that direct events of CHIP28 topogenesis. A signal sequence located within the first 52 residues initiated nascent chain translocation into the ER lumen. A stop transfer sequence located in the hydrophobic region from residues 90-120 terminated ongoing translocation. A second internal signal sequence, residues 155-186, reinitiated translocation of a COOH-terminal domain (residues 186-210) into the ER lumen. Integration of the nascent chain into the ER membrane occurred after synthesis of 107 residues and required the presence of two membrane-spanning regions. From this data, we propose a structural model for CHIP28 at the ER membrane in which four membrane- spanning alpha-helices form a central aqueous channel through the lipid bilayer and create a pathway for water transport.     

Signals and mechanisms for protein retention in the endoplasmic reticulum

After their co-translational insertion into the ER lumen or the ER membrane, most proteins are transported via the Golgi apparatus downstream on the secretory pathway while a few protein species are retained in the ER. Polypeptide retention in the ER is either signal-independent or depends on specific retention signals encoded by the primary sequence of the polypeptide. A first category, i.e. the newly synthesized polypeptides that are unable to reach their final conformation, are retained in the ER where this quality control generally results in their degradation. A second category, namely the ER-resident proteins escape the bulk flow of secretion due to the presence of a specific N- or C-terminal signal that interacts with integral membrane or soluble receptors. ER retention of soluble proteins mediated by either KDEL, HDEL or related sequences and membrane receptors has been relatively well characterized in plants. Recent efforts has been relatively well characterized in plants. Recent efforts have aimed at a characterization of the retention signal(s) of type I membrane proteins in the plant ER.     

Associations between subunit ectodomains promote T cell antigen receptor assembly and protect against degradation in the ER

The T cell antigen receptor (TCR) is an oligomeric protein complex made from at least six different integral membrane proteins (alpha beta gamma delta epsilon and zeta). The TCR is assembled in the ER of T cells, and correct assembly is required for transport to the cell surface. Single subunits and partial receptor complexes are retained in the ER where TCR alpha, beta, and CD3 delta chains are degraded selectively. The information required for the ER degradation of the TCR beta chain is confined to the membrane anchor of the protein (Wileman et al., 1990c; Bonifacino et al., 1990b). In this study we show that the rapid degradation of the TCR beta chain is inhibited when it assembles with single CD3 gamma, delta, or epsilon subunits in the ER, and have started to define the role played by transmembrane anchors, and receptor ectodomains, in the masking proteolytic targeting information. Acidic residues within the membrane spanning domains of CD3 subunits were essential for binding to the TCR beta chain. TCR beta chains and CD3 subunits therefore interact via transmembrane domains. However, when sites of binding were restricted to the membrane anchor of the TCR beta chain, stabilization by CD3 subunits was markedly reduced. Interactions between membrane spanning domains were not, therefore, sufficient for the protection of the beta chain from ER proteolysis. The presence of the C beta domain, containing the first 150 amino acids of the TCR ectodomain, greatly increased the stability of complexes formed in the ER. For assembly with CD3 epsilon, stability was further enhanced by the V beta amino acids. The results showed that the efficient neutralization of transmembrane proteolytic targeting information required associations between membrane spanning domains and the presence of receptor ectodomains. Interactions between receptor ectodomains may slow the dissociation of CD3 subunits from the beta chain and prolong the masking of transmembrane targeting information. In addition, the close proximity of TCR and CD3 ectodomains within the ER may provide steric protection from the action of proteases within the ER lumen.     

Signal-mediated retrieval of a membrane protein from the Golgi to the ER in yeast

The Saccharomyces cerevisiae Wbp1 protein is an endoplasmic reticulum (ER), type I transmembrane protein which contains a cytoplasmic dilysine (KKXX) motif. This motif has previously been shown to direct Golgi-to-ER retrieval of type I membrane proteins in mammalian cells (Jackson, M. R., T. Nilsson, and P. A. Peterson. 1993. J. Cell Biol. 121: 317-333). To analyze the role of this motif in yeast, we constructed a SUC2-WBP1 chimera consisting of the coding sequence for the normally secreted glycoprotein invertase fused to the coding sequence of the COOH terminus (including the transmembrane domain and 16-amino acid cytoplasmic tail) of Wbplp. Carbohydrate analysis of the invertase-Wbp1 fusion protein using mannose linkage-specific antiserum demonstrated that the fusion protein was efficiently modified by the early Golgi initial alpha 1,6 mannosyltransferase (Och1p). Subcellular fractionation revealed that > 90% of the alpha 1,6 mannose-modified fusion protein colocalized with the ER (Wbp1p) and not with the Golgi Och1p-containing compartment or other membrane fractions. Amino acid changes within the dily sine motif (KK-->QK, KQ, or QQ) did not change the kinetics of initial alpha 1,6 mannose modification of the fusion protein but did dramatically increase the rate of modification by more distal Golgi (elongating alpha 1,6 and alpha 1,3) mannosyltransferases. These mutant fusion proteins were then delivered directly from a late Golgi compartment to the vacuole, where they were proteolytically cleaved in a PEP4-dependent manner. While amino acids surrounding the dilysine motif played only a minor role in retention ability, mutations that altered the position of the lysines relative to the COOH terminus of the fusion protein also yielded a dramatic defect in ER retention. Collectively, our results indicate that the KKXX motif does not simply retain proteins in the ER but rather directs their rapid retrieval from a novel, Och1p-containing early Golgi compartment. Similar to observations in mammalian cells, it is the presence of two lysine residues at the appropriate COOH-terminal position which represents the most important features of this sorting determinant.     

Both a short hydrophobic domain and a carboxyl-terminal hydrophilic region are important for signal function in the Escherichia coli leader peptidase

Leader peptidase, typical of inner membrane proteins of Escherichia coli, does not have an amino-terminal leader sequence. This protein contains an internal signal peptide, residues 51-83, which is essential for assembly and remains as a membrane anchor domain. We have employed site-directed mutagenesis techniques to either delete residues within this domain or substitute a charged amino acid for one of these residues to determine the important properties of the internal signal. The deletion analysis showed that a very small apolar domain, residues 70-76, is essential for assembly, whereas residues that flank it are dispensable for its function. However, point mutations with charged amino acid residues within the polar sequence (residues 77-82) slow or abolish leader peptidase membrane assembly. Thus, a polar region, Arg-Ser-Phe-Ile-Tyr-Glu, is important for the signal peptide function of leader peptidase, unlike other signals identified thus far.