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.     

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.     

The signal peptide of the rotavirus glycoprotein VP7 is essential for its retention in the ER as an integral membrane protein

The rotavirus glycoprotein VP7 has a cleavable signal peptide and is normally resident as an integral membrane protein in the ER of infected cells. A gene was constructed in which the VP7 H2 signal peptide was replaced by one from influenza hemagglutinin. COS cells transfected with this gene produced VP7 with the correct amino terminus, but the protein was rapidly secreted. Uncleaved VP7 from either precursor was not detected in cells after brief pulse-labeling, suggesting that the signal peptide was not acting as a temporary anchor; rather, it exerted its effect despite rapid cleavage. By splicing the H2 signal peptide onto another reporter protein, the malaria S-antigen, we demonstrated that H2 was necessary, but not itself sufficient, for targeting and retention. We propose that an interaction between the cleaved signal peptide and other downstream sequences in VP7 is required for retention of this protein in the ER as an integral membrane polypeptide.     

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.     

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.     

Membrane binding and endoplasmic reticulum retention sequences of rotavirus VP7 are distinct: role of carboxy-terminal and other residues in membrane binding.

The sequences responsible for binding rotavirus glycoprotein VP7 to the membrane of the endoplasmic reticulum (ER) have not been identified. Here we show that the sequences which promote membrane binding in vitro are distinct from the N-terminal sequences which promote retention of VP7 in the ER in vivo. The role of the C-terminal region in membrane binding was also examined by using truncation mutants. Membrane binding in vitro was reduced but not abolished by removing up to 102 residues from the C terminus. The data suggest that the last 36 residues of VP7 may be present in the membrane or translocation pore, possibly with the C terminus protruding into the cytoplasm, since these residues contribute to, but do not account for, membrane binding. Surprisingly, modified forms of VP7 which are secreted from transfected cells showed the same membrane-binding properties in vitro as the protein retained in the ER membrane. Thus, secreted VP7 may not be present as a soluble polypeptide in the ER. A model to explain these results is presented. Previously published data are consistent with the idea that the highly conserved C terminus of nascent VP7 could have a cytoplasmic orientation which is important for assembly of mature virus particles.     

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.     

Diffusion in the endoplasmic reticulum of an aquaporin-2 mutant causing human nephrogenic diabetes insipidus

Mutations in the aquaporin-2 (AQP2) water channel cause the hereditary renal disease nephrogenic diabetes insipidus (NDI). The missense mutation AQP2-T126M causes human recessive NDI by retention at the endoplasmic reticulum (ER) of renal epithelial cells. To determine whether the ER retention of AQP2-T126M is due to relative immobilization in the ER, we measured by fluorescence recovery after photobleaching the intramembrane mobility of green fluorescent protein (GFP) chimeras containing human wild-type and mutant AQP2. In transfected LLC-PK1 renal epithelial cells, GFP-labeled AQP2-T126M was localized to the ER, and wild-type AQP2 to endosomes and the plasma membrane; both were localized to the ER after brefeldin A treatment. Photobleaching with image detection indicated that the GFP-AQP2 chimeras were freely mobile throughout the ER. Quantitative spot photobleaching revealed a diffusion-dependent irreversible process whose recovery depended on spot size and was abolished by paraformaldehyde fixation. In addition, a novel slow reversible fluorescence recovery (t(12) approximately 2 s) was characterized whose recovery was independent of spot size and not affected by fixation. AQP2 translational diffusion in the ER was not slowed by the T126M mutation; diffusion coefficients were (in cm(2)/s x 10(-)10) 2.6 +/- 0.5 (wild-type) and 3.0 +/- 0.4 (T126M). Much faster diffusion was found for a lipid probe (diOC(4)(3), 2.7 x 10(-)8 cm(2)/s) in the ER membrane and for unconjugated GFP in the aqueous ER lumen (6 x 10(-)8 cm(2)/s). ER diffusion of GFP-T126M was not significantly affected by up-regulation of molecular chaperones, cAMP activation, or actin filament disruption. ATP depletion by 2-deoxyglucose and azide resulted in comparable slowing/immobilization of wild-type and T126M AQP2. These results indicate that the ER retention of AQP2-T126M does not result from restricted or slowed mobility and suggest that the majority of AQP2-T126M is not aggregated or bound to slowly moving membrane proteins.     

Binding to membrane proteins within the endoplasmic reticulum cannot explain the retention of the glucose-regulated protein GRP78 in Xenopus oocytes.

We have studied the compartmentation and movement of the rat 78-kd glucose-regulated protein (GRP78) and other secretory and membrane proteins in Xenopus oocytes. Full length GRP78, normally found in the lumen of rat endoplasmic reticulum (ER), is localized to a membraneous compartment in oocytes and is not secreted. A truncated GRP78 lacking the C-terminal (KDEL) ER retention signal is secreted, although at a slow rate. When the synthesis of radioactive GRP78 is confined to a polar (animal or vegetal) region of the oocyte and the subsequent movement across the oocyte monitored, we find that both full-length and truncated GRP78 move at similar rates and only slightly slower than a secretory protein, chick ovalbumin. In contrast, a plasma membrane protein (influenza haemagglutinin) and two ER membrane proteins (rotavirus VP10 and a mutant haemagglutinin) remained confined to their site of synthesis. We conclude that the retention of GRP78 in the ER is not due to its tight binding to a membrane-bound receptor.     

Assembly of the herpes simplex virus capsid: requirement for the carboxyl-terminal twenty-five amino acids of the proteins encoded by the UL26 and UL26.5 genes.

Herpes simplex virus type 1 (HSV-1) intermediate capsids are composed of seven proteins, VP5, VP19C, VP21, VP22a, VP23, VP24, and VP26, and the genes that encode these proteins, UL19, UL38, UL26, UL26.5, UL18, UL26, and UL35, respectively. The UL26 gene encodes a protease that cleaves itself and the product of the UL26.5 gene at a site (M site) 25 amino acids from the C terminus of these two proteins. In addition, the protease cleaves itself at a second site (R site) between amino acids 247 and 248. Cleavage of the UL26 protein gives rise to the capsid proteins VP21 and VP24, and cleavage of the UL26.5 protein gives rise to the capsid protein VP22a. Previously we described the production of HSV-1 capsids in insect cells by infecting the cells with recombinant baculoviruses expressing the six capsid genes (D. R. Thomsen, L. L. Roof, and F. L. Homa, J. Virol. 68:2442-2457, 1994). Using this system, we demonstrated that the products of the UL26 and/or UL26.5 genes are required as scaffolds for assembly of HSV-1 capsids. To better understand the functions of the UL26 and UL26.5 proteins in capsid assembly, we constructed baculoviruses that expressed altered UL26 and UL26.5 proteins. The ability of the altered UL26 and UL26.5 proteins to support HSV-1 capsid assembly was then tested in insect cells. Among the specific mutations tested were (i) deletion of the C-terminal 25 amino acids from the proteins coded for by the UL26 and UL26.5 genes; (ii) mutation of His-61 of the UL26 protein, an amino acid required for protease activity; and (iii) mutation of the R cleavage site of the UL26 protein. Analysis of the capsids formed with wild-type and mutant proteins supports the following conclusions: (i) the C-terminal 25 amino acids of the UL26 and UL26.5 proteins are required for capsid assembly; (ii) the protease activity associated with the UL26 protein is not required for assembly of morphologically normal capsids; and (iii) the uncleaved forms of the UL26 and UL26.5 proteins are employed in assembly of 125-nm-diameter capsids; cleavage of these proteins occurs during or subsequent to capsid assembly. Finally, we carried out in vitro experiments in which the major capsid protein VP5 was mixed with wild-type or truncated UL26.5 protein and then precipitated with a VP5-specific monoclonal antibody.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins.

Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.     

Assembly of infectious Herpes simplex virus type 1 virions in the absence of full-length VP22

VP22, the 301-amino-acid phosphoprotein product of the herpes simplex virus type 1 (HSV-1) U(L)49 gene, is incorporated into the tegument during virus assembly. We previously showed that highly modified forms of VP22 are restricted to infected cell nuclei (L. E. Pomeranz and J. A. Blaho, J. Virol. 73:6769-6781, 1999). VP22 packaged into infectious virions appears undermodified, and nuclear- and virion-associated forms are easily differentiated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (J. A. Blaho, C. Mitchell, and B. Roizman, J. Biol. Chem. 269:17401-17410, 1994). As VP22 packaging-associated undermodification is unique among HSV-1 tegument proteins, we sought to determine the role of VP22 during viral replication. We now show the following. (i) VP22 modification occurs in the absence of other viral factors in cell lines which stably express its gene. (ii) RF177, a recombinant HSV-1 strain generated for this study, synthesizes only the amino-terminal 212 amino acids of VP22 (Delta212). (iii) Delta212 localizes to the nucleus and incorporates into virions during RF177 infection of Vero cells. Thus, the carboxy-terminal region is not required for nuclear localization of VP22. (iv) RF177 synthesizes the tegument proteins VP13/14, VP16, and VHS (virus host shutoff) and incorporates them into infectious virions as efficiently as wild-type virus. However, (v) the loss of VP22 in RF177 virus particles is compensated for by a redistribution of minor virion components. (vi) Mature RF177 virions are identical to wild-type particles based on electron microscopic analyses. (vii) Single-step growth kinetics of RF177 in Vero cells are essentially identical to those of wild-type virus. (viii) RF177 plaque size is reduced by nearly 40% compared to wild-type virus. Based on these results, we conclude that VP22 is not required for tegument formation, virion assembly/maturation, or productive HSV-1 replication, while the presence of full-length VP22 in the tegument is needed for efficient virus spread in Vero cell monolayers.     

Intracellular transport of rat serum albumin is altered by a genetically engineered deletion of the propeptide

Many secreted proteins are synthesized with aminoterminal propeptides which are removed prior to secretion. There is increasing interest in the physiological roles of these propeptides, especially as mediators of intracellular protein trafficking. To investigate whether or not the propeptide of serum albumin offers an advantage in albumin secretion, we used oligonucleotide-directed mutagenesis to delete the 18 base pairs which encode the propeptide from a cDNA gene for rat serum albumin (RSA), inserted the deleted gene into COS cells, and studied the secretion of the gene product (RSA delta pro). Quantitative enzyme-linked immunosorbent assay analysis of medium from transfected cells showed that RSA delta pro was secreted at about 64% of the level of RSA. Furthermore, pulse-chase protein labeling studies demonstrated that the rate of secretion for RSA delta pro was greatly decreased relative to RSA. Immunofluorescent analyses of transfected cells showed accumulation of RSA delta pro in the endoplasmic reticulum, suggesting that transport through and/or exit from the ER was affected. The electrophoretic migration of secreted and intracellular forms of RSA and RSA delta pro indicated that they were the same molecular weight, and a specific amino-terminal binding assay, using nickel 63, confirmed the absence and proper cleavage of the prepeptide. These findings demonstrate that transport of RSA delta pro through the secretion pathway is inhibited and that the inhibition is due to the absence of the propeptide.     

Structural requirements for high affinity ligand binding by estrogen receptors: a comparative analysis of truncated and full length estrogen receptors expressed in bacteria, yeast, and mammalian cells.

In order to better understand the structural requirements for effective high affinity binding of estrogens and antiestrogens by the human estrogen receptor (ER), a comparative study was undertaken in which we examined: 1) native ER from the MCF-7 ER-positive human breast cancer cell line; 2) full length ER expressed in yeast; 3) the ER hormone binding domain (amino acid residues 302-595) expressed in yeast; 4) a bacterially expressed protein A fusion product encoding a truncated ER (amino acid residues 240-595); and 5) a synthetic peptide encompassing amino acids 510-551 of the ER. The binding parameters studied included affinity, kinetics, structural specificity for ligands, and stability. Full length ER expressed in yeast was very similar to the MCF-7 ER in its affinity [dissociation constant (Kd), 0.35 +/- 0.05 nM], dissociation rate (t1/2, 3-4 h at 25 C), and structural specificity for both reversible and covalently attaching affinity ligands. While the truncated ER expressed in yeast was similar to MCF-7 ER in its specificity of ligand binding, it showed a slightly reduced affinity for estradiol (Kd, 1.00 +/- 0.17 nM). The bacterially expressed ER also had a lower affinity for estradiol (Kd, 1.49 +/- 0.16 nM), which may be due in part to an increase in the dissociation rate (t1/2, 0.5 h at 25 C). The attachment of covalent affinity ligands and structural specificity for a variety of reversible ligands was comparable in the bacterially expressed ER to that observed for the receptors expressed in MCF-7 cells and yeast.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of C-terminal truncations on trafficking of the yeast plasma membrane H+-ATPase

Within the large family of P-type cation-transporting ATPases, members differ in the number of C-terminal transmembrane helices, ranging from two in Cu2+-ATPases to six in H+-, Na+,K+-, Mg2+-, and Ca2+-ATPases. In this study, yeast Pma1 H+-ATPase has served as a model to examine the role of the C-terminal membrane domain in ATPase stability and targeting to the plasma membrane. Successive truncations were constructed from the middle of the major cytoplasmic loop to the middle of the extended cytoplasmic tail, adding back the C-terminal membrane-spanning helices one at a time. When the resulting constructs were expressed transiently in yeast, there was a steady increase in half-life from 70 min in Pma1 delta452 to 348 min in Pma1 delta901, but even the longest construct was considerably less stable than wild-type ATPase (t(1/2) = 11 h). Confocal immunofluorescence microscopy showed that 11 of 12 constructs were arrested in the endoplasmic reticulum and degraded in the proteasome. The only truncated ATPase that escaped the ER, Pma1 delta901, traveled slowly to the plasma membrane, where it hydrolyzed ATP and supported growth. Limited trypsinolysis showed Pma1 delta901 to be misfolded, however, resulting in premature delivery to the vacuole for degradation. As model substrates, this series of truncations affirms the importance of the entire C-terminal domain to yeast H+-ATPase biogenesis and defines a sequence element of 20 amino acids in the carboxyl tail that is critical to ER escape and trafficking to the plasma membrane.     

ER retention may play a role in sorting of the nuclear pore membrane protein POM121

Integral membrane proteins of the nuclear envelope (NE) are synthesized on the rough endoplasmic reticulum (ER) and following free diffusion in the continuous ER/NE membrane system are targeted to their proper destinations due to interactions of specific domains with other components of the NE. By studying the intracellular distribution and dynamics of a deletion mutant of an integral membrane protein of the nuclear pores, POM121, which lacks the pore-targeting domain, we investigated if ER retention plays a role in sorting of integral membrane proteins to the nuclear envelope. A nascent membrane protein lacking sorting determinants is believed to diffuse laterally in the continuous ER/NE lipid bilayer and expected to follow vesicular traffic to the plasma membrane. The GFP-tagged deletion mutant, POM121(1-129)-GFP, specifically distributed within the ER membrane, but was completely absent from the Golgi compartment and the plasma membrane. Experiments using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) demonstrated that despite having very high mobility within the whole ER network (D = 0.41 +/- 0.11 micro m(2)/s) POM121(1-129)-GFP was unable to exit the ER. It was also not detected in post-ER compartments of cells incubated at 15 degrees C. Taken together, these experiments show that amino acids 1-129 of POM121 are able to retain GFP in the ER membrane and suggest that this retention occurs by a direct mechanism rather than by a retrieval mechanism. Our data suggest that ER retention might be important for sorting of POM121 to the nuclear pores.     

Novel N-terminal amino acid sequence required for retention of a hepatitis B virus glycoprotein in the endoplasmic reticulum.

The preS1 surface glycoprotein of hepatitis B virus is targeted to the endoplasmic reticulum (ER) and is retained in this organelle when expressed in the absence of other viral gene products. The protein is also acylated at its N terminus with myristic acid. Sequences responsible for its ER retention have been identified through examination of mutants bearing lesions in the preS1 coding region. These studies reveal that such sequences map to the N terminus of the molecule, between residues 6 and 19. Molecules in which this region was present remained in the ER; those in which it had been deleted were secreted from the cell. Although all deletions which allowed efficient secretion also impaired acylation of the polypeptide, myristylation alone was not sufficient for ER retention: point mutations which eliminated myristylation did not lead to secretion. These data indicate that an essential element for ER retention resides in a 14-amino-acid sequence that is unrelated to previously described ER retention signals.     

Valosin-containing Protein-interacting Membrane Protein (VIMP) Links the Endoplasmic Reticulum with Microtubules in Concert with Cytoskeleton-linking Membrane Protein (CLIMP)-63

The distribution and morphology of the endoplasmic reticulum (ER) in mammalian cells depend on both dynamic and static interactions of ER membrane proteins with microtubules (MTs). Cytoskeleton-linking membrane protein (CLIMP)-63 is exclusively localized in sheet-like ER membranes, typical structures of the rough ER, and plays a pivotal role in the static interaction with MTs. Our previous study showed that the 42-kDa ER-residing form of syntaxin 5 (Syn5L) regulates ER structure through the interactions with both CLIMP-63 and MTs. Here, we extend our previous study and show that the valosin-containing protein/p97-interacting membrane protein (VIMP)/SelS is also a member of the family of proteins that shape the ER by interacting with MTs. Depletion of VIMP causes the spreading of the ER to the cell periphery and affects an MT-dependent process on the ER. Although VIMP can interact with CLIMP-63 and Syn5L, it does not interact with MT-binding ER proteins (such as Reep1) that shape the tubular smooth ER, suggesting that different sets of MT-binding ER proteins are used to organize different ER subdomains.