Transport of vesicular stomatitis virus G protein to the cell surface is signal mediated in polarized and nonpolarized cells

Current model propose that in nonpolarized cells, transport of plasma membrane proteins to the surface occurs by default. In contrast, compelling evidence indicates that in polarized epithelial cells, plasma membrane proteins are sorted in the TGN into at least two vectorial routes to apical and basolateral surface domains. Since both apical and basolateral proteins are also normally expressed by both polarized and nonpolarized cells, we explored here whether recently described basolateral sorting signals in the cytoplasmic domain of basolateral proteins are recognized and used for post TGN transport by nonpolarized cells. To this end, we compared the inhibitory effect of basolateral signal peptides on the cytosol-stimulated release of two basolateral and one apical marker in semi-intact fibroblasts (3T3), pituitary (GH3), and epithelial (MDCK) cells. A basolateral signal peptide (VSVGp) corresponding to the 29-amino acid cytoplasmic tail of vesicular stomatitis virus G protein (VSVG) inhibited with identical potency the vesicular release of VSVG from the TGN of all three cell lines. On the other hand, the VSVG peptide did not inhibit the vesicular release of HA in MDCK cells not of two polypeptide hormones (growth hormone and prolactin) in GH3 cells, whereas in 3T3 cells (influenza) hemagglutinin was inhibited, albeit with a 3x lower potency than VSVG. The results support the existence of a basolateral-like, signal-mediated constitutive pathway from TGN to plasma membrane in all three cell types, and suggest that an apical-like pathway may be present in fibroblast. The data support cargo protein involvement, not bulk flow, in the formation of post-TGN vesicles and predict the involvement of distinct cytosolic factors in the assembly of apical and basolateral transport vesicles.     

Rab8, a small GTPase involved in vesicular traffic between the TGN and the basolateral plasma membrane

Small GTP-binding proteins of the rab family have been implicated as regulators of membrane traffic along the biosynthetic and endocytic pathways in eukaryotic cells. We have investigated the localization and function of rab8, closely related to the yeast YPT1/SEC4 gene products. Confocal immunofluorescence microscopy and immunoelectron microscopy on filter-grown MDCK cells demonstrated that, rab8 was localized to the Golgi region, vesicular structures, and to the basolateral plasma membrane. Two-dimensional gel electrophoresis showed that rab8p was highly enriched in immuno-isolated basolateral vesicles carrying vesicular stomatitis virus-glycoprotein (VSV-G) but was absent from vesicles transporting the hemagglutinin protein (HA) of influenza virus to the apical cell surface. Using a cytosol dependent in vitro transport assay in permeabilized MDCK cells we studied the functional role of rab8 in biosynthetic membrane traffic. Transport of VSV-G from the TGN to the basolateral plasma membrane was found to be significantly inhibited by a peptide derived from the hypervariable COOH-terminal region of rab8, while transport of the influenza HA from the TGN to the apical surface and ER to Golgi transport were unaffected. We conclude that rab8 plays a role in membrane traffic from the TGN to the basolateral plasma membrane in MDCK cells.     

Annexin XIIIb: a novel epithelial specific annexin is implicated in vesicular traffic to the apical plasma membrane

The sorting of apical and basolateral proteins into vesicular carriers takes place in the trans-Golgi network (TGN) in MDCK cells. We have previously analyzed the protein composition of immunoisolated apical and basolateral transport vesicles and have now identified a component that is highly enriched in apical vesicles. Isolation of the encoding cDNA revealed that this protein, annexin XIIIb, is a new isoform of the epithelial specific annexin XIII sub-family which includes the previously described intestine-specific annexin (annexin XIIIa; Wice, B. M., and J. I. Gordon. 1992. J. Cell Biol. 116:405-422). Annexin XIIIb differs from annexin XIIIa in that it contains a unique insert of 41 amino acids in the NH2 terminus and is exclusively expressed in dog intestine and kidney. Immunofluorescence microscopy demonstrated that annexin XIIIb was localized to the apical plasma membrane and underlying punctate structures. Since annexins have been suggested to play a role in membrane-membrane interactions in exocytosis and endocytosis, we investigated whether annexin XIIIb is involved in delivery to the apical cell surface. To this aim we used permeabilized MDCK cells and a cytosol-dependent in vitro transport assay. Antibodies specific for annexin XIIIb significantly inhibited the transport of influenza virus hemagglutinin from the TGN to the apical plasma membrane while the transport of vesicular stomatitis virus glycoprotein to the basolateral cell surface was unaffected. We propose that annexin XIIIb plays a role in vesicular transport to the apical plasma membrane in MDCK cells.     

Caveolin-1 and -2 in the Exocytic Pathway of MDCK Cells

Abstract. We have studied the biosynthesis and transport of the endogenous caveolins in MDCK cells. We show that in addition to homooligomers of caveolin-1, heterooligomeric complexes of caveolin-1 and -2 are formed in the ER. The oligomers become larger, increasingly detergent insoluble, and phosphorylated on caveolin-2 during transport to the cell surface. In the TGN caveolin-1/-2 heterooligomers are sorted into basolateral vesicles, whereas larger caveolin-1 homooligomers are targeted to the apical side. Caveolin-1 is present on both the apical and basolateral plasma membrane, whereas caveolin-2 is enriched on the basolateral surface where caveolae are present. This suggests that caveolin-1 and -2 heterooligomers are involved in caveolar biogenesis in the basolateral plasma membrane. Anti–caveolin-1 antibodies inhibit the apical delivery of influenza virus hemagglutinin without affecting basolateral transport of vesicular stomatitis virus G protein. Thus, we suggest that caveolin-1 homooligomers play a role in apical transport.     

Basolateral protein transport in streptolysin O-permeabilized MDCK cells

We have reconstituted polarized protein transport in streptolysin O- permeabilized MDCK cells from the TGN to the basolateral surface and to the apical surface. These transport steps are dependent on temperature, energy and exogenously supplied cytosol. Using this in vitro system we show that a whole tail peptide (WT peptide) corresponding to the cytoplasmic tail of a basolaterally sorted protein, the vesicular stomatitis virus glycoprotein (VSV G) inhibits the TGN to basolateral transport but does not affect any other transport step. Inhibition of VSV G transport to basolateral surface by WT peptide did not result in missorting of the protein to the apical surface. Mutation of the single tyrosine residue in the WT peptide reduced its inhibitory potency four- to fivefold. These results suggest that the VSV G tail physically interacts with a component of the sorting machinery. Using a cross- linking approach, we have identified proteins that associate with the cytoplasmic tail domain of VSV G. One of these polypeptides, Tin-2 (Tail interacting protein-2), associates with VSV G in the TGN, the site of protein sorting, but not in the ER nor at the cell surface. Tin- 2 does not associate with apically targeted hemagglutinin. WT peptide that inhibited the basolateral transport of VSV G also inhibited the association of Tin-2 with VSV G. Together, these properties make Tin-2 a candidate basolateral sorter. The results demonstrate the usefulness of the SLO-permeabilized cell system in dissecting the sorting machinery.     

Analysis of the role of p200-containing vesicles in post-Golgi traffic.

p200 is a cytoplasmic protein that associates with vesicles budding from the trans-golgi network (TGN). The protein was identified by a monoclonal antibody AD7. We have used this antibody to analyze whether p200 functions in exocytic transport from the TGN to the apical or basolateral plasma membrane in Madin-Darby canine kidney cells. We found that transport of the viral marker proteins, influenza hemagglutinin (HA) to the apical surface or vesicular stomatitis virus glycoprotein (VSV G) to the basolateral surface in streptolysin O-permeabilized cells was not affected when p200 was depleted from both the membranes and the cytosol. When vesicles isolated from perforated cells were analyzed by equilibrium density gradient centrifugation, the p200 immunoreactive membranes did not comigrate with either the apical vesicle marker HA or the basolateral vesicle marker VSV G. Immunoelectron microscopy of perforated and double-labeled cells showed that the p200 positive vesicular profiles were not labeled by antibodies to HA or VSV G when the viral proteins were accumulated in the TGN. Furthermore, the p200-decorated vesicles were more electron dense than those labeled with the viral antibodies. Together, these results suggest that p200 does not function in the transport pathways that carry HA from the TGN to the apical surface or VSV G from the TGN to the basolateral surface.     

Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts

Rab8 is a small Ras-like GTPase that regulates polarized membrane transport to the basolateral membrane in epithelial cells and to the dendrites in neurons. It has recently been demonstrated that fibroblasts sort newly synthesized proteins into two different pathways for delivery to the cell surface that are equivalent to the apical and the basolateral post-Golgi routes in epithelial cells (Yoshimori, T., P. Keller, M.G. Roth, and K. Simons. 1996. J. Cell Biol. 133:247-256). To determine the role of Rab8 in fibroblasts, we used both transient expression systems and stable cell lines expressing mutant or wild-type (wt) Rab8. A dramatic change in cell morphology occurred in BHK cells expressing both the wt Rab8 and the activated form of the GTPase, the Rab8Q67L mutant. These cells formed processes as a result of a reorganization of both their actin filaments and microtubules. Newly synthesized vesicular stomatitis virus G glycoprotein, a basolateral marker protein in MDCK cells, was preferentially delivered into these cell outgrowths. Based on these findings, we propose that Rab8 provides a link between the machinery responsible for the formation of cell protrusions and polarized biosynthetic membrane traffic.     

Alteration of the cytoplasmic domain of the membrane-spanning glycoprotein p62 of Semliki Forest virus does not affect its polar distribution in established lines of Madin-Darby canine kidney cells

Expression of the Semliki Forest virus p62/E2 protein was studied in the polarized epithelial cell line Madin-Darby canine kidney (MDCK). After infection this transmembrane protein, together with the other spike subunit E1, accumulates at the basolateral surface of MDCK cells (Fuller, S. D., C.-H. von Bonsdorff, and K. Simons, 1985, EMBO (Eur. Mol. Biol. Organ.) J., 4:2475-2485). The cDNAs encoding truncated forms of the protein were used to stably transform MDCK cells to examine the role of subunit oligomerization (E1-E2) and the cytoplasmic domain of p62/E2 in directed transport to the basolateral surface. The biochemical characteristics and polarity of the expressed proteins were studied using cell monolayers grown on nitrocellulose filters. A wild- type form of p62/E2, in the absence of E1, and two forms having either 15 or 3 of the wild-type 31-amino acid carboxyl cytoplasmic domain were all localized to the basolateral surface. These results indicate that the cytoplasmic domain of E2 does not contain the information essential for directed transport to the plasma membrane, and imply that this information resides in either the lumenal and/or membrane-spanning segments of this transmembrane protein.     

Different membrane anchors allow the Semliki Forest virus spike subunit E2 to reach the cell surface.

The Semliki Forest virus spike subunit E2, a membrane-spanning protein, was transported to the plasma membrane in BHK cells after its carboxy terminus, including the intramembranous and cytoplasmic portions, was replaced by respective fragments of either the vesicular stomatitis virus glycoprotein or the fowl plague virus hemagglutinin. The hybrid proteins were constructed by cDNA fusion. Upon a transient expression they could be localized at the cell surface by immunofluorescence with specific antibodies directed against any of the protein fragments.     

Mutations in the Middle of the Transmembrane Domain Reverse the Polarity of Transport of the Influenza Virus Hemagglutinin in MDCK Epithelial Cells

The composition of the plasma membrane domains of epithelial cells is maintained by biosynthetic pathways that can sort both proteins and lipids into transport vesicles destined for either the apical or basolateral surface. In MDCK cells, the influenza virus hemagglutinin is sorted in the trans-Golgi network into detergent-insoluble, glycosphingolipid-enriched membrane domains that are proposed to be necessary for sorting hemagglutinin to the apical cell surface. Site- directed mutagenesis of the hemagglutinin transmembrane domain was used to test this proposal. The region of the transmembrane domain required for apical transport included the residues most conserved among hemagglutinin subtypes. Several mutants were found to enter detergent-insoluble membranes but were not properly sorted. Replacement of transmembrane residues 520 and 521 with alanines converted the 2A520 mutant hemagglutinin into a basolateral protein. Depleting cell cholesterol reduced the ability of wild-type hemagglutinin to partition into detergent-insoluble membranes but had no effect on apical or basolateral sorting. In contrast, cholesterol depletion allowed random transport of the 2A520 mutant. The mutant appeared to lack sorting information but was prevented from reaching the apical surface when detergent-insoluble membranes were present. Apical sorting of hemagglutinin may require binding of either protein or lipids at the middle of the transmembrane domain and this normally occurs in detergent-insoluble membrane domains. Entry into these domains appears necessary, but not sufficient, for apical sorting.     

Transport of influenza HA from the trans-Golgi network to the apical surface of MDCK cells permeabilized in their basolateral plasma membranes: energy dependence and involvement of GTP-binding proteins

A procedure employing streptolysin O to effect the selective permeabilization of either the apical or basolateral plasma membrane domains of MDCK cell monolayers grown on a filter support was developed which permeabilizes the entire monolayer, leaves the opposite cell surface domain intact, and does not abolish the integrity of the tight junctions. This procedure renders the cell interior accessible to exogenous macromolecules and impermeant reagents, permitting the examination of their effects on membrane protein transport to the intact surface. The last stages of the transport of the influenza virus hemagglutinin (HA) to the apical surface were studied in pulse-labeled, virus-infected MDCK cells that were incubated at 19.5 degrees C for 90 min to accumulate newly synthesized HA in the trans-Golgi network (TGN), before raising the temperature to 35 degrees C to allow synchronized transport to the plasma membrane. In cells permeabilized immediately after the cold block, 50% of the intracellular HA molecules were subsequently delivered to the apical surface. This transport was dependent on the presence of an exogenous ATP supply and was markedly inhibited by the addition of GTP-gamma-S at the time of permeabilization. On the other hand, the GTP analogue had no effect when it was added to cells that, after the cold block, were incubated for 15 min at 35 degrees C before permeabilization, even though at this time most HA molecules were still intracellular and their appearance at the cell surface was largely dependent on exogenous ATP. These findings indicate that GTP-binding proteins are involved in the constitutive process that effects vesicular transport from the TGN to the plasma membrane and that they are charged early in this process. Transport of HA to the cell surface could be made dependent on the addition of exogenous cytosol when, after permeabilization, cells were washed to remove endogenous cytosolic components. This opens the way towards the identification of cell components that mediate the sorting of apical and basolateral membrane components in the TGN and their polarized delivery to the cell surface.     

TGN38 recycles basolaterally in polarized Madin-Darby canine kidney cells.

Sorting of newly synthesized plasma membrane proteins to the apical or basolateral surface domains of polarized cells is currently thought to take place within the trans-Golgi network (TGN). To explore the relationship between protein localization to the TGN and sorting to the plasma membrane in polarized epithelial cells, we have expressed constructs encoding the TGN marker, TGN38, in Madin-Darby canine kidney (MDCK) cells. We report that TGN38 is predominantly localized to the TGN of these cells and recycles via the basolateral membrane. Analyses of the distribution of Tac-TGN38 chimeric proteins in MDCK cells suggest that the cytoplasmic domain of TGN38 has information leading to both TGN localization and cycling through the basolateral surface. Mutations of the cytoplasmic domain that disrupt TGN localization also lead to nonpolarized delivery of the chimeric proteins to both surface domains. These results demonstrate an apparent equivalence of basolateral and TGN localization determinants and support an evolutionary relationship between TGN and plasma membrane sorting processes.     

Cholesterol Is Required for Surface Transport of Influenza Virus Hemagglutinin

Transport from the TGN to the basolateral surface involves a rab/N-ethylmaleimide–sensitive fusion protein (NSF)/soluble NSF attachment protein (SNAP)/SNAP receptor (SNARE) mechanism. Apical transport instead is thought to be mediated by detergent-insoluble sphingolipid–cholesterol rafts. By reducing the cholesterol level of living cells by 60–70% with lovastatin and methyl-β-cyclodextrin, we show that the TGN-to-surface transport of the apical marker protein influenza virus hemagglutinin was slowed down, whereas the transport of the basolateral marker vesicular stomatitis virus glycoprotein as well as the ER-to-Golgi transport of both membrane proteins was not affected. Reduction of transport of hemagglutinin was accompanied by increased solubility in the detergent Triton X-100 and by significant missorting of hemagglutinin to the basolateral membrane. In addition, depletion of cellular cholesterol by lovastatin and methyl-β-cyclodextrin led to missorting of the apical secretory glycoprotein gp-80, suggesting that gp-80 uses a raft-dependent mechanism for apical sorting. Our data provide for the first time direct evidence for the functional significance of cholesterol in the sorting of apical membrane proteins as well as of apically secreted glycoproteins.     

The MAL proteolipid is necessary for the overall apical delivery of membrane proteins in the polarized epithelial Madin-Darby canine kidney and fischer rat thyroid cell lines

The MAL proteolipid has been recently demonstrated as being necessary for correct apical sorting of the transmembrane influenza virus hemagglutinin (HA) in Madin-Darby canine kidney (MDCK) cells. The fact that, in contrast to MDCK cells, Fischer rat thyroid (FRT) cells target the majority of glycosylphosphatidylinositol (GPI)-anchored proteins to the basolateral membrane provides us with the opportunity to determine the role of MAL in apical transport of membrane proteins under conditions in which the majority of GPI-anchored proteins are (MDCK cells) or are not (FRT cells) targeted to the apical surface. Using an antisense oligonucleotide-based strategy to deplete endogenous MAL, we have observed that correct transport of apical transmembrane proteins associated (HA) or not (exogenous neurotrophin receptor and endogenous dipeptidyl peptidase IV) with lipid rafts, as well as that of the bulk of endogenous apical membrane, takes place in FRT cells by a pathway that requires normal MAL levels. Even transport of placental alkaline phosphatase, a GPI-anchored protein that is targeted apically in FRT cells, was dependent on normal MAL levels. Similarly, in addition to the reported effect of MAL on HA transport, depletion of MAL in MDCK cells caused a dramatic reduction in the apical delivery of the GPI-anchored gD1-DAF protein, neurotrophin receptor, and the bulk of membrane proteins. These results suggest that MAL is necessary for the overall apical transport of membrane proteins in polarized MDCK and FRT cells.     

Detergent insoluble microdomains are not involved in transcytosis of polymeric Ig receptor in FRT and MDCK cells

In polarized epithelial cells, sorting of proteins and lipids to the apical or basolateral domain of the plasma membrane can occur via direct or indirect (transcytotic) pathways from the trans Golgi network (TGN). The 'rafts' hypothesis postulates that the key event for direct apical sorting of some transmembrane proteins and the majority of GPI-anchored proteins depends on their association with glycosphingolipid and cholesterol enriched microdomains (rafts). However, the mechanism of indirect sorting to the apical membrane is not clear. The polyimmunoglobulin receptor (pIgR) is one of the best studied proteins that follow the transcytotic pathway. It is normally delivered from the TGN to the basolateral surface of polarized Madin–Darby Canine Kidney (MDCK) cells from where it transports dIgA or dIgM to the apical surface. We have studied the intracellular trafficking of pIgR in Fischer rat thyroid cells (FRT), and have investigated the sorting machinery involved in transcytosis of this receptor in both FRT and MDCK cells. We found that, in contrast with MDCK cells, a significant amount (∼30%) of pIgR reaches the apical surface by a direct pathway. Furthermore, in both cell lines it does not associate with Triton X-100-insoluble microdomains, suggesting that at least in these cells 'rafts' are not involved in basolateral to apical transcytosis.     

Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins.

Polarized expression of most epithelial plasma membrane proteins is achieved by selective transport from the Golgi apparatus or from endosomes to a specific cell surface domain. In Madin-Darby canine kidney (MDCK) cells, basolateral sorting generally depends on distinct cytoplasmic targeting determinants. Inactivation of these signals often resulted in apical expression, suggesting that apical transport of transmembrane proteins occurs either by default or is mediated by widely distributed characteristics of membrane glycoproteins. We tested the hypothesis of N-linked carbohydrates acting as apical targeting signals using three different membrane proteins. The first two are normally not glycosylated and the third one is a glycoprotein. In all three cases, N-linked carbohydrates were clearly able to mediate apical targeting and transport. Cell surface transport of proteins containing cytoplasmic basolateral targeting determinants was not significantly affected by N-linked sugars. In the absence of glycosylation and a basolateral sorting signal, the reporter proteins accumulated in the Golgi complex of MDCK as well as CHO cells, indicating that efficient transport from the Golgi apparatus to the cell surface is signal-mediated in polarized and non-polarized cells.     

Microtubule perturbation inhibits intracellular transport of an apical membrane glycoprotein in a substrate-dependent manner in polarized Madin-Darby canine kidney epithelial cells.

The effects of microtubule perturbation on the transport of two different viral glycoproteins were examined in infected Madin-Darby canine kidney (MDCK) cells grown on both permeable and solid substrata. Quantitative biochemical analysis showed that the microtubule-depolymerizing drug nocodazole inhibited arrival of influenza hemagglutinin on the apical plasma membrane in MDCK cells grown on both substrata. In contrast, the microtubule-stabilizing drug taxol inhibited apical appearance of hemagglutinin only when MDCK cells were grown on permeable substrata. On the basis of hemagglutinin mobility on sodium dodecyl sulfate gels and its sensitivity to endo H, it was evident that nocodazole and taxol arrested hemagglutinin at different intracellular sites. Neither drug caused a significant increase in the amount of hemagglutinin detected on the basolateral plasma membrane domain. In addition, neither drug had any noticeable effect on the transport of the vesicular stomatitis virus (VSV)-G protein to the basolateral surface. These results shed light on previous conflicting reports using this model system and support the hypothesis that microtubules play a role in the delivery of membrane glycoproteins to the apical, but not the basolateral, domain of epithelial cells.     

Sorting of an apical plasma membrane glycoprotein occurs before it reaches the cell surface in cultured epithelial cells

In Madin-Darby canine kidney (MDCK) cells (a polarized epithelial cell line) infected with influenza virus, the hemagglutinin behaves as an apical plasma membrane glycoprotein. To determine biochemically the domain on the plasma membrane, apical or basolateral, where newly synthesized hemagglutinin first appears, cells were cultured on Millipore filters to make both cell surface domains independently accessible. Hemagglutinin in virus-infected cells was pulse-labeled, chased, and detected on the plasma membrane with a sensitive trypsin assay. Under all conditions tested, newly made hemagglutinin appeared simultaneously on both domains, with the bulk found in the apical membrane. When trypsin was continuously present on the basolateral surface during the chase, little hemagglutinin was cleaved relative to the amount transported apically. In addition, specific antibodies against the hemagglutinin placed basolaterally had no effect on transport to the apical domain. These observations suggested that most newly synthesized hemagglutinin does not transiently appear on the basolateral surface but rather is delivered directly to the apical surface in amounts that account for its final polarized distribution.     

Cell surface influenza haemagglutinin can mediate infection by other animal viruses.

We have used filter-grown Madin-Darby canine kidney (MDCK) cells to explore the mechanism by which influenza virus facilitates secondary virus infection. Vesicular stomatitis virus (VSV) and Semliki Forest virus (SFV) infect only through the basolateral surface of these polarized epithelial cells and not through the apical surface. Prior infection with influenza virus rendered the cell susceptible to infection by VSV or SFV through either surface. The presence of both a permissive and a restrictive surface for virus entry in the same cell allowed us to determine how the influenza infection enhanced the subsequent infection of a second virus. Biochemical and morphological evidence showed that influenza haemagglutinin on the apical surface serves as a receptor for the superinfecting virus by binding to its sialic acid-bearing envelope proteins. Influenza virus also facilitates secondary virus infection in non-epithelial cells; baby hamster kidney cells (BHK-21), which are normally resistant to infection by the coronavirus (mouse hepatitis virus MHV-A59), could be infected via the haemagglutinin-sialic acid interaction. Facilitation of secondary virus infection requires only the sialic acid-binding properties of the haemagglutinin since the uncleaved haemagglutinin could also mediate virus entry.     

Caveolin-1 is not essential for biosynthetic apical membrane transport

Caveolin-1 has been implicated in apical transport of glycosylphosphatidylinositol (GPI)-anchored proteins and influenza virus hemagglutinin (HA). Here we have studied the role of caveolin-1 in apical membrane transport by generating caveolin-1-deficient Madin-Darby canine kidney (MDCK) cells using retrovirus-mediated RNA interference. The caveolin-1 knockdown (cav1-KD) MDCK cells were devoid of caveolae. In addition, caveolin-2 was retained in the Golgi apparatus in cav1-KD MDCK cells. However, we found no significant alterations in the apical transport kinetics of GPI-anchored proteins or HA upon depletion of caveolin-1. Similar results were obtained using embryonic fibroblasts from caveolin-1-knockout mice. Thus, we conclude that caveolin-1 does not play a major role in lipid raft-mediated biosynthetic membrane trafficking.