Targeting and retention of Golgi membrane proteins.

Recent cloning of genes encoding membrane proteins of the Golgi complex has allowed investigation of protein targeting to this organelle. Targeting signals have been identified in three glycosyltransferases, a viral envelope protein and several proteins of the trans-Golgi network. Interestingly, the targeting signals for membrane proteins of the Golgi stacks seem to be contained in transmembrane domains. Information in the cytoplasmic tails is required for the targeting of trans-Golgi network proteins. Mechanisms involving both retention and retrieval have been invoked.

Acquisition of cytotoxic T lymphocyte-specific carbohydrate differentiation antigens.

Cytotoxic T cell (CTL)-specific activation antigens, termed CT determinants, have been detected by monoclonal antibodies (mAb) that inhibit CTL function. At the cell surface, the CT antigens are associated with the T200 glycoproteins and two other proteins of Mr 140,000 and 85,000 and are present on a secreted protein, gp155. Periodate treatment followed by binding analysis and immunoprecipitation experiments using tunicamycin-treated cells indicated that carbohydrate is necessary for CT antigen expression. Furthermore, gp155 is secreted in the presence of tunicamycin while retaining the CT antigens, and the CT determinants are added late in T200 biosynthesis, suggesting that the CT glycans are O-linked. Finally, interleukin 2 was shown to dramatically influence the expression of the CT mAb-reactive oligosaccharides present at the CTL cell surface.

Golgi retention signals: do membranes hold the key?

The diverse forms and functions of cellular organelles are, presumably, a consequence of their particular molecular compositions. The generation and maintenance of this diversity is achieved by the targeting of newly synthesized proteins to specific locations and their subsequent retention there. Sequences that retain proteins in the endoplasmic reticulum (ER) have been identified at the C-termini of resident ER proteins, where they are readily accessible to potential receptors. By contrast, recent results have demonstrated that retention of proteins in the Golgi complex involves sequences located within transmembrane domains. This suggests the novel possibility that the membrane composition of the Golgi complex plays a role in retention of resident Golgi proteins.

Ceramide accumulation uncovers a cycling pathway for the cis-Golgi network marker, infectious bronchitis virus M protein.

The M glycoprotein from the avian coronavirus, infectious bronchitis virus (IBV), contains information for localization to the cis-Golgi network in its first transmembrane domain. We hypothesize that localization to the Golgi complex may depend in part on specific interactions between protein transmembrane domains and membrane lipids. Because the site of sphingolipid synthesis overlaps the localization of IBV M, we asked whether perturbation of sphingolipids affected localization of IBV M. Short-term treatment with two inhibitors of sphingolipid synthesis had no effect on localization of IBV M or other Golgi markers. Thus, ongoing synthesis of these lipids was not required for proper localization. Surprisingly, a third inhibitor, d,l-threo-1-phenyl-2-decanoylamino-3-morpholino- 1-propanol (PDMP), shifted the steady-state distribution of IBV M from the Golgi complex to the ER. This effect was rapid and reversible and was also observed for ERGIC-53 but not for Golgi stack proteins. At the concentration of PDMP used, conversion of ceramide into both glucosylceramide and sphingomyelin was inhibited. Pretreatment with upstream inhibitors partially reversed the effects of PDMP, suggesting that ceramide accumulation mediates the PDMP-induced alterations. Indeed, an increase in cellular ceramide was measured in PDMP-treated cells. We propose that IBV M is at least in part localized by retrieval mechanisms. Further, ceramide accumulation reveals this cycle by upsetting the balance of anterograde and retrograde traffic and/ or disrupting retention by altering bilayer dynamics.

Cytoplasmic domains of cellular and viral integral membrane proteins substitute for the cytoplasmic domain of the vesicular stomatitis virus glycoprotein in transport to the plasma membrane.

Oligonucleotide-directed mutagenesis was used to construct chimeric cDNAs that encode the extracellular and transmembrane domains of the vesicular stomatitis virus glycoprotein (G) linked to the cytoplasmic domain of either the immunoglobulin mu membrane heavy chain, the hemagglutinin glycoprotein of influenza virus, or the small glycoprotein (p23) of infectious bronchitis virus. Biochemical analyses and immunofluorescence microscopy demonstrated that these hybrid genes were correctly expressed in eukaryotic cells and that the hybrid proteins were transported to the plasma membrane. The rate of transport to the Golgi complex of G protein with an immunoglobulin mu membrane cytoplasmic domain was approximately sixfold slower than G protein with its normal cytoplasmic domain. However, this rate was virtually identical to the rate of transport of micron heavy chain molecules measured in the B cell line WEHI 231. The rate of transport of G protein with a hemagglutinin cytoplasmic domain was threefold slower than wild type G protein and G protein with a p23 cytoplasmic domain, which were transported at similar rates. The combined results underscore the importance of the amino acid sequence in the cytoplasmic domain for efficient transport of G protein to the cell surface. Also, normal cytoplasmic domains from other transmembrane glycoproteins can substitute for the G protein cytoplasmic domain in transport of G protein to the plasma membrane. The method of constructing precise hybrid proteins described here will be useful in defining functions of specific domains of viral and cellular integral membrane proteins.

A specific transmembrane domain of a coronavirus E1 glycoprotein is required for its retention in the Golgi region.

The E1 glycoprotein of the avian coronavirus infectious bronchitis virus contains a short, glycosylated amino-terminal domain, three membrane-spanning domains, and a long carboxy-terminal cytoplasmic domain. We show that E1 expressed from cDNA is targeted to the Golgi region, as it is in infected cells. E1 proteins with precise deletions of the first and second or the second and third membrane-spanning domains were glycosylated, thus suggesting that either the first or third transmembrane domain can function as an internal signal sequence. The mutant protein with only the first transmembrane domain accumulated intracellularly like the wild-type protein, but the mutant protein with only the third transmembrane domain was transported to the cell surface. This result suggests that information specifying accumulation in the Golgi region resides in the first transmembrane domain, and provides the first example of an intracellular membrane protein that is transported to the plasma membrane after deletion of a specific domain.

A Golgi retention signal in a membrane-spanning domain of coronavirus E1 protein.

The E1 glycoprotein from an avian coronavirus is a model protein for studying retention in the Golgi complex. In animal cells expressing the protein from cDNA, the E1 protein is targeted to cis Golgi cisternae (Machamer, C. E., S. A. Mentone, J. K. Rose, and M. G. Farquhar. 1990. Proc. Natl. Acad. Sci. USA. 87:6944-6948). We show that the first of the three membrane-spanning domains of the E1 protein can retain two different plasma membrane proteins in the Golgi region of transfected cells. Both the vesicular stomatitis virus G protein and the alpha-subunit of human chorionic gonadotropin (anchored to the membrane by fusion with the G protein membrane-spanning domain and cytoplasmic tail) were retained in the Golgi region of transfected cells when their single membrane-spanning domains were replaced with the first membrane-spanning domain from E1. Single amino acid substitutions in this sequence released retention of the chimeric G protein, as well as a mutant E1 protein which lacks the second and third membrane-spanning domains. The important feature of the retention sequence appears to be the uncharged polar residues which line one face of a predicted alpha helix. This is the first retention signal to be defined for a resident Golgi protein. The fact that it is present in a membrane-spanning domain suggests a novel mechanism of retention in which the membrane composition of the Golgi complex plays an instrumental role in retaining its resident proteins.

Oligomerization of a membrane protein correlates with its retention in the Golgi complex.

The first membrane-spanning domain (m1) of the M glycoprotein of avian coronavirus (formerly called E1) is sufficient to retain this protein in the cis-Golgi. When the membrane-spanning domain of a protein which is efficiently delivered to the plasma membrane (VSV G protein) is replaced with m1, the resulting chimera (Gm1) is retained in the Golgi (Swift, A. M., and C. E. Machamer. 1991. J. Cell Biol. 115:19-30). When assayed in sucrose gradients, we observed that Gm1 formed a large oligomer, and that much of this oligomer was SDS resistant and stayed near the top of the stacking gel of an SDS-polyacrylamide gel. The unusual stability of the oligomer allowed it to be detected easily. Gm1 mutants with single amino acid substitutions in the m1 domain that were retained in the Golgi complex formed SDS-resistant oligomers, whereas mutants that were rapidly released to the plasma membrane did not. Oligomerization was not detected immediately after synthesis of Gm1, but occurred gradually with a lag of approximately 10 min, suggesting that it is not merely aggregation of misfolded proteins. Furthermore, oligomerization did not occur under several conditions that block ER to Golgi transport. The lumenal domain was not required for oligomerization since another chimera (alpha m1G), where the lumenal domain of Gm1 was replaced by the alpha subunit of human chorionic gonadotropin, also formed an SDS-resistant oligomer, and was able to form hetero-oligomers with Gm1 as revealed by coprecipitation experiments. SDS resistance was conferred by the cytoplasmic tail of VSV G, because proteolytic digestion of the tail in microsomes containing Gm1 oligomers resulted in loss of SDS resistance, although the protease-treated material continued to migrate as a large oligomer on sucrose gradients. Interestingly, treatment of cells with cytochalasin D blocked formation of SDS-resistant (but not SDS-sensitive) oligomers. Our data suggest that SDS-resistant oligomers form as newly synthesized molecules of Gm1 arrive at the Golgi complex and may interact (directly or indirectly) with an actin-based cytoskeletal matrix. The oligomerization of Gm1 and other resident proteins could serve as a mechanism for their retention in the Golgi complex.

Caspase-2 is localized at the Golgi complex and cleaves golgin-160 during apoptosis.

Caspases are an extended family of cysteine proteases that play critical roles in apoptosis. Animals deficient in caspases-2 or -3, which share very similar tetrapeptide cleavage specificities, exhibit very different phenotypes, suggesting that the unique features of individual caspases may account for distinct regulation and specialized functions. Recent studies demonstrate that unique apoptotic stimuli are transduced by distinct proteolytic pathways, with multiple components of the proteolytic machinery clustering at distinct subcellular sites. We demonstrate here that, in addition to its nuclear distribution, caspase-2 is localized to the Golgi complex, where it cleaves golgin-160 at a unique site not susceptible to cleavage by other caspases with very similar tetrapeptide specificities. Early cleavage at this site precedes cleavage at distal sites by other caspases. Prevention of cleavage at the unique caspase-2 site delays disintegration of the Golgi complex after delivery of a pro-apoptotic signal. We propose that the Golgi complex, like mitochondria, senses and integrates unique local conditions, and transduces pro-apoptotic signals through local caspases, which regulate local effectors.

Assembly of vaccinia virus: role of the intermediate compartment between the endoplasmic reticulum and the Golgi stacks.

Vaccinia virus, the prototype of the Poxviridae, is a large DNA virus which replicates in the cytoplasm of the host cell. The assembly pathway of vaccinia virus displays several unique features, such as the production of two structurally distinct, infectious forms. One of these, termed intracellular naked virus (INV), remains cells associated while the other, termed extracellular enveloped virus (EEV), is released from the cell. In addition, it has long been believed that INVs acquire their lipid envelopes by a unique example of de novo membrane biogenesis. To examine the structure and assembly of vaccinia virus we have used immunoelectron microscopy using antibodies to proteins of different subcellular compartments as well as a phospholipid analysis of purified INV and EEV. Our data are not consistent with the de novo model of viral membrane synthesis but rather argue that the vaccinia virus DNA becomes enwrapped by a membrane cisterna derived from the intermediate compartment between the ER and the Golgi stacks, thus acquiring two membranes in one step. Phospholipid analysis of purified INV supports its derivation from an early biosynthetic compartment. This unique assembly process is repeated once more when the INV becomes enwrapped by an additional membrane cisterna, in agreement with earlier reports. The available data suggest that after fusion between the outer envelope and the plasma membrane, mature EEV is released from the cell.

A single N-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus G protein to the cell surface.

We investigated the role of glycosylation in intracellular transport and cell surface expression of the vesicular stomatitis virus glycoprotein (G) in cells expressing G protein from cloned cDNA. The individual contributions of the two asparagine-linked glycans of G protein to cell surface expression were assessed by site-directed mutagenesis of the coding sequence to eliminate one or the other or both of the glycosylation sites. One oligosaccharide at either position was sufficient for cell surface expression of G protein in transfected cells, and the rates of oligosaccharide processing were similar to the rate observed for wild-type protein. However, the nonglycosylated G protein synthesized when both glycosylation sites were eliminated did not reach the cell surface. This protein did appear to reach a Golgi-like region, as determined by indirect immunofluorescence microscopy, however, and was modified with palmitic acid. It was also apparently not subject to increased proteolytic breakdown.

Heterogeneous distribution of the unusual phospholipid semilysobisphosphatidic acid through the Golgi complex.

To investigate the distribution of lipids through the Golgi complex, we analyzed the envelopes of several viruses that assemble in different subcompartments of the Golgi, as well as subcellular fractions. Our results indicate that each Golgi subcompartment has a distinct phospholipid composition due mainly to differences in the relative amounts of semilysobisphosphatidic acid (SLBPA), sphingomyelin, phosphatidylserine, and phosphatidylinositol. Interestingly, SLBPA is enriched in the adjacent Golgi networks compared with the Golgi stack, and this enrichment varies with cell type. The heterogeneous distribution of SLBPA through the Golgi complex suggests it may play an important role in the structure and/or function of this organelle.

Efficient export of the vesicular stomatitis virus G protein from the endoplasmic reticulum requires a signal in the cytoplasmic tail that includes both tyrosine-based and di-acidic motifs.

The vesicular stomatitis virus (VSV) G protein is a model transmembrane glycoprotein that has been extensively used to study the exocytotic pathway. A signal in the cytoplasmic tail of VSV G (DxE or Asp-x-Glu, where x is any amino acid) was recently proposed to mediate efficient export of the protein from the endoplasmic reticulum (ER). In this study, we show that the DxE motif only partially accounts for efficient ER exit of VSV G. We have identified a six-amino-acid signal, which includes the previously identified Asp and Glu residues, that is required for efficient exit of VSV G from the ER. This six-residue signal also includes the targeting sequence YxxO (where x is any amino acid and O is a bulky, hydrophobic residue) implicated in several different sorting pathways. The only defect in VSV G proteins with mutations in the six-residue signal is slow exit from the ER; folding and oligomerization in the ER are normal, and the mutants eventually reach the plasma membrane. Addition of this six-residue motif to an inefficiently transported reporter protein is sufficient to confer an enhanced ER export rate. The signal we have identified is highly conserved among divergent VSV G proteins, and we suggest this reflects the importance of this motif in the evolution of VSV G as a proficient exocytic protein.

Cholesterol-independent targeting of Golgi membrane proteins in insect cells.

Distinct lipid compositions of intracellular organelles could provide a physical basis for targeting of membrane proteins, particularly where transmembrane domains have been shown to play a role. We tested the possibility that cholesterol is required for targeting of membrane proteins to the Golgi complex. We used insect cells for our studies because they are cholesterol auxotrophs and can be depleted of cholesterol by growth in delipidated serum. We found that two well-characterized mammalian Golgi proteins were targeted to the Golgi region of Aedes albopictus cells, both in the presence and absence of cellular cholesterol. Our results imply that a cholesterol gradient through the secretory pathway is not required for membrane protein targeting to the Golgi complex, at least in insect cells.

Retention of a cis Golgi protein requires polar residues on one face of a predicted alpha-helix in the transmembrane domain.

The first membrane-spanning domain (m1) of the model cis Golgi protein M (formerly called E1) from the avian coronavirus infectious bronchitis virus is required for targeting to the Golgi complex. When inserted in place of the membrane-spanning domain of a plasma membrane protein (vesicular stomatitis virus G protein), the chimeric protein ("Gm1") is retained in the Golgi complex of transfected cells. To determine the precise features of the m1 domain responsible for Golgi targeting, we produced single amino acid substitutions in m1 and analyzed their effects on localization of Gm1. Expression at the plasma membrane was used as the criterion for loss of Golgi retention. Rates of oligosaccharide processing were used as a measure of rate and efficiency of transport through the Golgi complex. We identified four uncharged polar residues that are critical for Golgi retention of Gm1 (Asn465, Thr469, Thr476, and Gln480). These residues line one face of a predicted alpha-helix. Interestingly, when the m1 domain of the homologous M protein from mouse hepatitis virus is inserted into the G protein reporter, the chimeric protein is not efficiently retained in the Golgi complex, but transported to the cell surface. Although it possesses three of the four residues we identified as important in the avian m1 sequence, other residues in the membrane-spanning domain from the mouse protein must prevent efficient recognition of the polar face within the lipid bilayer of the cis Golgi.

Analysis of monoclonal antibodies reactive with human class II beta chains by two-dimensional electrophoresis and Western blotting.

We have used the Western blotting technique to examine B lymphoblastoid cell line (B-LCL) membrane proteins separated by two-dimensional gel electrophoresis specifically to analyze the binding patterns of monoclonal antibodies to separated HLA class II antigen beta (beta) subunits. The B-LCL LG-10 (homozygous for DR7), in which at least two sets of class II molecules can be distinguished on the basis of different electrophoretic mobilities, was examined with five monoclonal antibodies which detect monomorphic determinants. Four of the antibodies reacted with only DR beta subunits, while one antibody, XD5.A11, reacted with DR and with additional beta chains. Examination of two polymorphic monoclonal antibodies, SFR3-DR5, specific for HLA-DR5, and SFR3-PI.1, which reacts with a determinant absent from DR3 and DR7 homozygous lines, showed that both bind beta subunits from Swei, a DR5 homozygous line. Purification of a subpopulation of Swei class II molecules using an SFR3-PI.1 affinity column showed that the determinants recognized by SFR3-DR5, SFR3-PI.1, and a monomorphic monoclonal antibody reactive only with HLA-DR beta subunits of LG-10, reacted with identical beta subunits. Additional class II antigen subunits reactive with XD5.A11 were nonreactive with the polymorphic antibodies and the HLA-DR-specific monomorphic monoclonal antibody.

Elimination of mycoplasma from human B-lymphoblastoid cell lines.

Intraperitoneal passage of human B-lymphoblastoid cell lines in nude mice was examined as a means of mycoplasma eradication. Recovery of viable cells from the mice was facilitated by immediate plating on feeder layers of human foreskin fibroblasts. In all cases, nude mouse passage for as little as 5 days was totally effective in removing all contaminating mycoplasma.

Influence of new glycosylation sites on expression of the vesicular stomatitis virus G protein at the plasma membrane.

In this report, we have asked whether asparagine-linked oligosaccharides added to new sites in the polypeptide backbone of a model plasma membrane glycoprotein, the vesicular stomatitis virus G protein, can promote its intracellular transport. We modified the coding sequence of G protein lacking the two normal consensus sites for glycosylation by oligonucleotide-directed mutagenesis to create new consensus sites. The expression of the mutant proteins was then analyzed in transfected cells. Six of the eight new sites which were introduced were glycosylated, and an oligosaccharide at two of these new sites promoted transport of G protein which lacked the two normal sites. However, the efficiency of this process was reduced compared to the wild-type protein or to the proteins with only one oligosaccharide at either of the normal sites. In addition, an oligosaccharide at two of the other new sites caused inhibition of transport of the wild-type G protein. The data in this and the following report suggest that carbohydrate plays an indirect role in the intracellular transport of G protein.

Vesicular stomatitis virus G proteins with altered glycosylation sites display temperature-sensitive intracellular transport and are subject to aberrant intermolecular disulfide bonding.

In this report we have extended our studies on a panel of vesicular stomatitis virus G proteins with altered glycosylation sites. These mutant proteins were generated by oligonucleotide-directed mutagenesis of the coding sequence to create new consensus sites for asparagine-linked oligosaccharide addition. We report that the intracellular transport of most of the mutant proteins is temperature-sensitive, implying a polypeptide folding step is affected. In addition, we find that the nonglycosylated G protein and those mutant proteins which lack oligosaccharides at the normal positions are subject to aberrant intermolecular disulfide bonding, leading to the accumulation of large complexes in the endoplasmic reticulum. These results imply that carbohydrate plays an indirect role in the intracellular transport of G protein.