Primary sequence domains required for the retention of rotavirus VP7 in the endoplasmic reticulum

Rotavirus VP7 is a membrane-associated protein of the endoplasmic reticulum (ER). It is the product of rotavirus gene 9 which potentially encodes a protein of 326 amino acids that contains two amino terminal hydrophobic domains, h1 and h2, each preceded by an initiation codon. Comparison of the size of products derived from altered genes containing coding sequences for both h1 and h2 with those lacking the h1 sequence ('dhl' mutants), indicates that initiation takes place at M30 immediately preceding h2 (residues F32 to L48) and that h2 is cleaved, confirming the studies of others (Stirzaker, S.C., P.L. Whitfeld, D.L. Christie, A.R. Bellamy, and G.W. Both. 1987. J. Cell Biol. 105:2897-2903). Our previous work had shown that deletions in the carboxy end of h2, extending to amino acid 61 in the open reading frame, resulted in secretion of VP7. The region from amino acid number 51-61, present in wild-type VP7 but missing in the secreted mutant delta 47-61, was thus implicated to have a role in ER retention. To test this, a series of chimeric genes were constructed by fusing the first 63 codons of wild-type VP7, delta 1-14 or delta 51-61/dhl, to the mouse salivary alpha-amylase gene, a secretory protein, such that the fusion junction was located at the exact mature terminus of amylase. The chimeric proteins VP7(63)/amylase, delta 1-14(63)/amylase and delta 51-61(63)/dhl/amylase were secreted when expressed in cells and the h2 domain was cleaved when mRNA was translated in vitro. These results imply that the sequence 51-61 is necessary but not sufficient for ER retention. When a second series of VP7/amylase chimera were constructed extending the VP7 contribution to amino acid 111, the product expressed by delta 1-14(111)/amylase was not secreted whereas that of delta 47-61(111)/amylase was. Significantly, the intracellular delta 1-14(111)/amylase product exhibited an amylase enzymatic specific activity that was similar to that of the wild-type amylase product. We conclude that two regions of VP7 mediate its retention in the ER, the first lies within the sequence 51-61 and the second within the sequence 62-111, which contains the glycosylation site for VP7. Both regions are necessary for retention, though neither is sufficient alone.     

Retention by the endoplasmic reticulum of rotavirus VP7 is controlled by three adjacent amino-terminal residues.

The rotavirus outer capsid glycoprotein, VP7, is an endoplasmic reticulum (ER) membrane-associated glycoprotein in both infected and transfected cells. It was previously demonstrated in this laboratory and by others that both the cleaved signal sequence (H2) and the first NH2-terminal 61 amino acids of VP7 are sufficient and necessary for ER retention of this molecule. Using site-specific mutagenesis and transfection techniques, we show that residues Ile-9, Thr-10, and Gly-11 were specifically necessary for ER retention. These results further define the ER retention sequence of VP7 and demonstrate that conservative changes, apparently innocuous in only three adjacent amino acids, can lead to major solubility and compartmentalization changes. It was found that placement of the first 31 mature NH2-terminal residues of VP7, in addition to the cleaved ER translocation signal sequence, was sufficient to retain the enzymatically active chimeric alpha-amylase in the ER; this enzyme is normally secreted. Deletions of the residues Ile-9, Thr-10, and Gly-11 within the amylase chimera containing 31 VP7 amino acids resulted in secretion of enzymatically active protein. It was also observed that the residues of VP7 presented in certain chimeras were able to abolish alpha-amylase enzymatic activity. These chimeras are presumably misfolded since it was demonstrated by pulse-chase experiments that these molecules are degraded in the ER. We surmise that a favorable conformation is necessary for retention since ER retention and activity of the chimeras depend on the primary sequence context.     

Two forms of VP7 are involved in assembly of SA11 rotavirus in endoplasmic reticulum

Two pools of the glycoprotein VP7 were detected in the endoplasmic reticulum (ER) of SA11 rotavirus-infected cells. One portion of the newly synthesized protein with VP3 composed the virus outer capsid, while the rest remained associated with the membrane. The two populations could be separated biochemically by fluorocarbon extraction or by immunological methods which used two classes of antibodies. A monoclonal antibody with neutralizing activity recognized VP7 only as displayed on intact virus particles, while a polyclonal antiserum precipitated predominantly the unassembled ER form of the protein and precipitated virus-assembled VP7 poorly. Virus-associated VP7 was localized by immunofluorescence to small punctate structures, presumably corresponding to accumulated virus particles, and to regions of the ER surrounding viroplasmic inclusions, whereas the membrane-associated molecules were distributed in an arborizing reticular pattern throughout the ER. VP3 and the nonstructural glycoprotein NCVP5 displayed a localization similar to that of virus-associated VP7. Intracellular virus particles were isolated from infected cells to determine the kinetics of assembly of VP7 and of the other structural proteins into virions. It was found that incorporation of the inner capsid proteins into single-shelled particles occurred rapidly, while VP7 and VP3 appeared in mature double-shelled particles with a lag time of 10 to 15 min. In addition, the alpha-mannosidase processing kinetics of virus-associated VP7 oligosaccharides showed a 15-min lag compared with that of the membrane-associated form, suggesting that the latter is the precursor to virion VP7. This lag may represent the time required for virus budding and outer capsid assembly.     

Location of sequences within rotavirus SA11 glycoprotein VP7 which direct it to the endoplasmic reticulum.

The Simian 11 rotavirus glycoprotein VP7 is directed to the endoplasmic reticulum (ER) of the cell and retained as an integral membrane protein. The gene coding for VP7 predicts two potential initiation codons, each of which precedes a hydrophobic region of amino acids (H1 and H2) with the characteristics of a signal peptide. Using the techniques of gene mutagenesis and expression, we have determined that either hydrophobic domain alone can direct VP7 to the ER. A protein lacking both hydrophobic regions was not transported to the ER. Some polypeptides were directed across the ER membrane and then into the secretory pathway of the cell. For a variant retaining only the H1 domain, secretion was cleavage dependent, since an amino acid change which prevented cleavage also stopped secretion. However, secretion of two other deletion mutants lacking H1 and expressing truncated H2 domains was unaffected by this mutation, suggesting that these proteins were secreted without cleavage of their NH2-terminal hydrophobic regions or secreted after cleavage at a site(s) not predicted by current knowledge.     

Sequences in rotavirus glycoprotein VP7 that mediate delayed translocation and retention of the protein in the endoplasmic reticulum

Glycosylation and translocation of the simian rotavirus protein VP7, a resident ER protein, does not occur co-translationally in vivo. In pulse-chase experiments in COS cells, nonglycosylated VP7 was still detectable after a 25-min chase period, although the single glycosylation site was only 18 residues beyond the signal peptide cleavage site. After labeling, glycosylated and nonglycosylated VP7 was recovered in microsomes but the latter was sensitive to trypsin (i.e., the nascent protein became membrane associated) but most of it entered the ER posttranslationally because of a rate-limiting step early in translocation. In contrast with the simian protein, bovine VP7 was glycosylated and translocated rapidly. Thus, delayed translocation per se was not required for retention of VP7 in the ER. By constructing hybrid proteins, it was further shown that the signal peptide together with residues 64-111 of the simian protein caused delayed translocation. The same sequences were also necessary and sufficient for retention of simian VP7 in the ER. The data are consistent with the idea that certain proteins are inserted into the ER membrane in a loop configuration.     

Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein

Rotavirus, a non-enveloped reovirus, buds into the rough endoplasmic reticulum and transiently acquires a membrane. The structural glycoprotein, VP7, a 38-kD integral membrane protein of the endoplasmic reticulum (ER), presumably transfers to virus in this process. The gene for VP7 potentially encodes a protein of 326 amino acids which has two tandem hydrophobic domains at the NH2-terminal, each preceded by an in-frame ATG codon. A series of deletion mutants constructed from a full-length cDNA clone of the Simian 11 rotavirus VP7 gene were expressed in COS 7 cells. Products from wild-type, and mutants which did not affect the second hydrophobic domain of VP7, were localized by immunofluorescence to elements of the ER only. However, deletions affecting the second hydrophobic domain (mutants 42-61, 43-61, 47-61) showed immunofluorescent localization of VP7 which coincided with that of wheat germ agglutinin, indicating transport to the Golgi apparatus. Immunoprecipitable wild-type protein, or an altered protein lacking the first hydrophobic sequence, remained intracellular and endo-beta-N-acetylglucosaminidase H sensitive. In contrast, products of mutants 42-61, 43-61, and 47-61 were transported from the ER, and secreted. Glycosylation of the secreted molecules was inhibited by tunicamycin, resistant to endo-beta-N-acetylglucosaminidase H digestion and therefore of the N-linked complex type. An unglycosylated version of VP7 was also secreted. We suggest that the second hydrophobic domain contributes to a positive signal for ER location and a membrane anchor function. Secretion of the mutant glycoprotein implies that transport can be constitutive with the destination being dictated by an overriding compartmentalization signal.     

Two proline residues are essential in the calcium-binding activity of rotavirus VP7 outer capsid protein.

Rotavirus maturation and stability of the outer capsid are calcium-dependent processes. It has been shown previously that the concentration of Ca2+-solubilizing outer capsid proteins from rotavirus particles is dependent on the virus strain. This property of viral particles has been associated with the gene coding for VP7 (gene 9). In this study the correlation between VP7 and resistance to low [Ca2+] was confirmed by analyzing the origin of gene 9 from reassortant viruses prepared under the selective pressure of low [Ca2+]. After chemical mutagenesis, we selected mutant viruses of the bovine strain RF that are more resistant to low [Ca2+]. The genes coding for the VP7 proteins of these independent mutants have been sequenced. Sequence analysis confirmed that these mutants are independent and revealed that all mutant VP7 proteins have proline 75 changed to leucine and have an outer capsid that solubilized at low [Ca2+]. The mutation of proline 279 to serine is found in all but two mutants. The phenotype of mutants having a single proline change can be distinguished from the phenotype of mutants having two proline changes. Sequence analysis showed that position 75 is in a region (amino acids 65 to 78) of great variability and that proline 75 is present in most of the bovine strains. In contrast, proline 279 is in a conserved region and is conserved in all the VP7 sequences in data banks. This region is rich in oxygenated residues that are correctly allocated in the metal-coordinating positions of the Ca2+-binding EF-hand structure pattern, suggesting that this region is important in the Ca2+ binding of VP7.     

The carboxy-terminal 10 amino acid residues of cytochrome b5 are necessary for its targeting to the endoplasmic reticulum.

Cytochrome b5 is an integral membrane protein located on the outer surface of the endoplasmic reticulum (ER). This cytochrome is considered to be synthesized on free ribosomes and to be inserted post-translationally into the ER membrane, without participation of a signal recognition particle. To elucidate the signal responsible for targeting of cytochrome b5 to the ER membrane in vivo, DNAs encoding various derivatives of the cytochrome were constructed and introduced into cultured mammalian COS cells, and the subcellular distributions of the derivatives expressed in the cells were then analyzed. The deletion of more than 11 amino acid residues at the carboxy-terminal end of cytochrome b5 abolished the targeting and anchoring of the cytochrome to the ER membrane. Fusion proteins consisting of the carboxy-terminal 10 amino acid residues of cytochrome b5 and passenger proteins with the hydrophobic portion could be localized in the ER membrane. Thus, the last 10 amino acid residues of cytochrome b5 carry information necessary for the cytochrome to be targeted to the ER membrane.     

The immunogenicity of VP7, a rotavirus antigen resident in the endoplasmic reticulum, is enhanced by cell surface expression.

The glycoprotein VP7, the major serotype antigen of rotaviruses, is localized to the endoplasmic reticulum (ER) of the cell, where it is retained as a membrane-associated protein before assembly into mature virus particles. Wild-type VP7 expressed by a recombinant vaccinia virus was also located internally and was poorly antigenic. Using recombinant techniques, a correctly processed, secreted form of VP7 (S.C. Stirzaker and G.W. Both, Cell 56:741-747, 1989) was modified by addition to its C terminus of the membrane anchor and cytoplasmic domains from the influenza virus hemagglutinin. The hybrid protein was directed to the surface of cells, where it was anchored in the plasma membrane. When expressed in mice and rabbits by a recombinant vaccinia virus, the surface-anchored antigen stimulated a level of rotavirus-specific antibodies that was greater than 100-fold above the level induced by wild-type VP7. T-cell responses to the novel antigen were also elevated in comparison with the wild-type, intracellular protein. Cell surface anchoring may provide a strategy to increase the immunogenicity of intracellular antigens from other parasites and viruses.     

Characterization of the carboxyl-terminal sequences responsible for protein retention in the endoplasmic reticulum.

The COOH-terminal sequence KDEL has been shown to be essential for the retention of several proteins in the lumen of the endoplasmic reticulum (Munro S., and Pelham, H. R. B. (1987) Cell 48, 899-907; Pelham, H. R. B. (1988) EMBO J. 7, 913-918; Mazzarella; R. A., Srinivasan, M., Haugejorden, S. M., and Green, M. (1990) J. Biol. Chem. 265, 1092-1101). We have previously demonstrated that variants to the KDEL retention signal, particularly at the initial two positions of the tetrapeptide, can be made without affecting its ability to direct intracellular retention when appended to the neuropeptide Y precursor (pro-NPY) (Andres, D. A., Dickerson, I. M., and Dixon, J. E. (1990) J. Biol. Chem. 265, 5952-5955). To further investigate the nature of the KDEL retention signal, oligonucleotide-directed mutagenesis and transfection was used to generate stable mouse anterior pituitary AtT-20 cell lines expressing pro-NPY mutants with variants of the KDEL sequence added to their direct carboxyl terminus. Analyses of dibasic processing and indirect immunofluorescent microscopy of AtT-20 subclones were consistent with the retention of the pro-NPY mutants bearing the COOH-terminal extensions QDEL, KEDL, or KDEI within the endoplasmic reticulum. A change in the final amino acid of the tetrapeptide from Leu to Val abolished retention completely, and the peptide hormone was processed and secreted. These results indicate that only a limited number of conservative changes can be made to the final two positions of the tetrapeptide without abolishing activity and suggest a highly specific interaction of the retention signal and the KDEL receptor.     

SSR alpha and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane

GTP phosphorylation of rough microsomes in vitro is limited to four integral membrane proteins. Two of these, a phosphoprotein (pp90) and a phosphoglycoprotein (pgp35) were purified as a complex with two nonphosphorylated membrane glycoproteins, gp25H and gp25L. The authenticity of this complex was confirmed using two different purification procedures and by coimmunoprecipitation. By immunofluorescence a reticulated cytoplasmic network was revealed for the proteins which was similar to that for Louvard et al. (Louvard, D., Reggio, H., and Warren, G. (1982) J. Cell Biol. 92, 92-107) marker antisera which also recognized purified pp90 on immunoblots. Amino acid sequencing of peptides derived from pgp35 identified this protein as SSR alpha, an endoplasmic reticulum constituent as identified by cross-linking of translocating nascent chains (G?rlich, D, Prehn, S., Hartmann, E., Herz, J., Otto, A., Kraft, R., Wiedmann, M., Knespel, S., Dobberstein, B., and Rapoport, T. A. (1990) J. Cell Biol. 111, 2283-2294). The sequence of gp25H was determined from cDNA clones and was identical with SSR beta identified by G?rlich et al. (1990) as being tightly bound to SSR alpha. Sequencing of gp25L revealed no similarity of the deduced sequence with other proteins. However, pp90 revealed a high degree of sequence identity with the Ca(2+)-binding protein, calreticulin. 45Ca2+ overlay studies indicated that pp90 bound Ca2+ and the name calnexin is proposed. Surprisingly, pgp25 (SSR alpha) also bound Ca2+ although gp25H (SSR beta) and gp25L did not. Triton X-114 partitioning of the integral membrane proteins of rough microsomes suggested that pgp35 (SSR alpha) and calnexin were major Ca(2+)-binding proteins of the endoplasmic reticulum membrane. We propose that the function of the complex is to regulate Ca(2+)-dependent retention mechanisms for luminal proteins of the endoplasmic reticulum.     

Overlapping signal sequences control nuclear localization and endoplasmic reticulum retention of GRP58

Glucose-regulated GRP58 has shown clinical applications to endoplasmic reticulum (ER) stress and cancer. GRP58 is localized in the cytosol, endoplasmic reticulum (ER) and nucleus. Twenty-four amino acids at the N-terminal hydrophobic region are known to target GRP58 to ER for synthesis at the ER membrane and translocation into the ER lumen. In addition, GRP58 contains putative nuclear localization (494KPKKKKK500) and ER retention (502QEDL505) signals. However, the role of these signals in nuclear import and ER retention of GRP58 remains unknown. Present studies investigated the signals that control nuclear localization and ER retention of GRP58. Deletion/mutation of nuclear localization signal (NLS) abrogated nuclear import of GRP58. NLS attached to EGFP localized EGFP in the nucleus. However, deletion/mutation of putative ER retention signal alone did not alter ER retention of GRP58. Interestingly, a combined deletion/mutation of NLS and ER retention signals blocked the GRP58 retention in the ER. These results concluded that overlapping NLS and ER retention signal sequences regulate nuclear localization and ER retention of GRP58.     

Thiol-mediated protein retention in the endoplasmic reticulum: the role of ERp44

Formation of disulfide bonds, an essential step for the maturation and exit of secretory proteins from the endoplasmic reticulum (ER), is controlled by specific ER-resident enzymes. A pivotal element in this process is Ero1alpha, an oxidoreductin that lacks known ER retention motifs. Here we show that ERp44 mediates Ero1alpha ER localization through the formation of reversible mixed disulfides. ERp44 also prevents the secretion of an unassembled cargo protein with unpaired cysteines. We conclude that ERp44 is a key element in thiol-mediated retention. It might also favour the maturation of disulfide-linked oligomeric proteins and their quality control.     

Peripheral membrane proteins of sarcoplasmic and endoplasmic reticulum. Comparison of carboxyl-terminal amino acid sequences

Peripheral endoplasmic reticulum membrane proteins residing in the lumen of the endoplasmic reticulum occupy the same space as other secreted proteins. The presence of a four amino acid salvage or retention signal (KDEL-COOH = Lys-Asp-Glu-Leu-COOH) at the carboxyl-terminal end of peripheral membrane proteins has been shown to represent a signal or an essential part of a signal for their retention within the endoplasmic reticulum membrane. In heart and skeletal muscle, a number of sarcoplasmic reticulum proteins have recently been identified which are peripheral membrane proteins. The high-affinity calcium-binding protein (55 kilodaltons (kDa] appears to conform to the above described mechanisms and contains the KDEL carboxyl-terminal tetrapeptide. Thyroid hormone binding protein is present in the sarcoplasmic reticulum, in addition to its endoplasmic reticulum location, and has a modified but related tetrapeptide sequence (RDEL = Arg-Asp-Glu-Leu), which also probably functions as the retention signal. Calsequestrin and a 53-kDa glycoprotein, two other peripheral membrane proteins residing in the lumen of the sarcoplasmic reticulum, do not contain the KDEL retention signal. The sarcoplasmic reticulum may have developed a unique retention mechanism(s) for these muscle-specific proteins.     

Static retention of the lumenal monotopic membrane protein torsinA in the endoplasmic reticulum

TorsinA is a membrane-associated enzyme in the endoplasmic reticulum (ER) lumen that is mutated in DYT1 dystonia. How it remains in the ER has been unclear. We report that a hydrophobic N-terminal domain (NTD) directs static retention of torsinA within the ER by excluding it from ER exit sites, as has been previously reported for short transmembrane domains (TMDs). We show that despite the NTD's physicochemical similarity to TMDs, it does not traverse the membrane, defining torsinA as a lumenal monotopic membrane protein and requiring a new paradigm to explain retention. ER retention and membrane association are perturbed by a subset of nonconservative mutations to the NTD, suggesting that a helical structure with defined orientation in the membrane is required. TorsinA preferentially enriches in ER sheets, as might be expected for a lumenal monotopic membrane protein. We propose that the principle of membrane-based protein sorting extends to monotopic membrane proteins, and identify other proteins including the monotopic lumenal enzyme cyclooxygenase 1 (prostaglandin H synthase 1) that share this mechanism of retention with torsinA.     

Role of potentially charged transmembrane residues in targeting proteins for retention and degradation within the endoplasmic reticulum.

The selective breakdown of newly synthesized proteins retained within the endoplasmic reticulum (ER) is probably mediated by the specific recognition of structural features of protein substrates by components of a degradative system. Within the alpha chain of the multisubunit T-cell antigen receptor (TCR) complex, a transmembrane sequence containing two basic amino acid residues has been shown to act as a determinant for retention and rapid degradation in the ER. We now demonstrate that single basic or acidic amino acid residues can cause targeting for retention and degradation in the ER when placed within the transmembrane domain of an integral membrane protein normally destined for the cell surface. The effect of such potentially charged residues is dependent on their relative position within the transmembrane sequence and on the nature of the amino acid side chains. The phenotypic changes induced by potentially charged transmembrane residues occur without apparent alterations of the global folding or transmembrane topology of the mutant proteins. These observations test the hypothesis that potentially charged residues within transmembrane domains can provide the basis for a motif for ER degradation and explain the selective breakdown of some proteins retained within the ER.     

Integration of cytochrome b5 into endoplasmic reticulum membrane: participation of carboxy-terminal portion of the transmembrane domain

Integration of cytochrome b(5) (b5), a tail-anchored protein located in the endoplasmic reticulum (ER) membrane, into the membrane was studied. Mutation of three amino acids, -Leu-Met-Tyr, at the carboxy-terminal end of the transmembrane segment of b5 to alanines resulted in localization of the mutated protein, b5LMY/AAA, in the cytosol as well as in the ER membrane. When an N-glycosylation site was introduced at the carboxy-terminal end of b5LMY/AAA, a substantial amount of the glycosylated form of the mutant protein was recovered in the cytosol fraction. A portion of the mutant protein recovered in the ER was released from the membrane by incubation with the cytosol fraction, but no further release was observed in the second incubation, suggesting that b5 is present in two different states, loosely-bound and firmly-integrated forms, in the ER membrane. These results suggest that b5 is integrated into the ER membrane via the loosely bound state, in which the carboxy-terminal end of the molecule is inserted into the luminal side of the vesicle but is easily translocated back to the cytosol, and that the three amino acids are important for conversion of the loosely-bound state to the firmly-integrated state.     

Two distinct sequences control the targeting and anchoring of the mouse P450 1A1 into the yeast endoplasmic reticulum membrane.

We previously expressed mouse P450 1A1 in the yeast S. cerevisiae. In the present study, I describe experiments in which several deletions in the 5' end of the corresponding cDNA were created. The truncated forms were then expressed in yeast cells. Studies of microsomes obtained from transformed yeast show that the signal-sequence is not required in vivo for the integration of mouse P450 1A1 into the endoplasmic reticulum membrane. In addition, the cytochrome deleted for its hydrophobic signal-sequence appears to be enzymatically functional. These results strongly argue for the existence of a second determinant of membrane targeting and binding.