Secretion of recombinant proteins in yeast can be affected by their improper folding in the endoplasmic reticulum and subsequent elimination of the misfolded molecules via the endoplasmic reticulum associated protein degradation pathway. Recombinant proteins can also be degraded by the vacuolar protease complex. Human urokinase type plasminogen activator (uPA) is poorly secreted by yeast but the mechanisms interfering with its secretion are largely unknown.
We show that in Hansenula polymorpha overexpression worsens uPA secretion and stimulates its intracellular aggregation. The absence of the Golgi modifications in accumulated uPA suggests that aggregation occurs within the endoplasmic reticulum. Deletion analysis has shown that the N-terminal domains were responsible for poor uPA secretion and propensity to aggregate. Mutation abolishing N-glycosylation decreased the efficiency of uPA secretion and increased its aggregation degree. Retention of uPA in the endoplasmic reticulum stimulates its aggregation.
The data obtained demonstrate that defect of uPA secretion in yeast is related to its retention in the endoplasmic reticulum. Accumulation of uPA within the endoplasmic reticulum disturbs its proper folding and leads to formation of high molecular weight aggregates.
Protein trafficking is achieved by a bidirectional vesicle flow between the
various compartments of the eukaryotic cell. COPII coated vesicles mediate
anterograde protein transport from the endoplasmic reticulum to the Golgi
apparatus, whereas retrograde Golgi-to-endoplasmic reticulum vesicles use the
COPI coat. Inactivation of COPI vesicle formation in conditional
sec21 (γ-COP) mutants rapidly blocks transport of certain
proteins along the early secretory pathway. We have identified the integral
membrane protein Mst27p as a strong suppressor of sec21-3 and
ret1-1 mutants. A C-terminal KKXX motif of Mst27p that allows direct
binding to the COPI complex is crucial for its suppression ability. Mst27p and
its homolog Yar033w (Mst28p) are part of the same complex. Both proteins
contain cytoplasmic exposed C termini that have the ability to interact
directly with COPI and COPII coat complexes. Site-specific mutations of the
COPI binding domain abolished suppression of the sec21 mutants. Our
results indicate that overexpression of MST27 provides an increased
number of coat binding sites on membranes of the early secretory pathway and
thereby promotes vesicle formation. As a consequence, the amount of cargo that
can bind COPI might be important for the regulation of the vesicle flow in the
early secretory pathway.
Retrograde trafficking from the Golgi to the endoplasmic reticulum (ER) depends on the formation of vesicles coated with the multiprotein complex COPI. In Saccharomyces cerevisiae ubiquitinated derivatives of several COPI subunits have been identified. The importance of this modification of COPI proteins is unknown. With the exception of the Sec27 protein (β’COP) neither the ubiquitin ligase responsible for ubiquitination of COPI subunits nor the importance of this modification are known. Here we find that the ubiquitin ligase mutation, rsp5-1, has a negative effect that is additive with ret1-1 and sec28Δ mutations, in genes encoding α- and ε-COP, respectively. The double ret1-1 rsp5-1 mutant is also more severely defective in the Golgi-to-ER trafficking compared to the single ret1-1, secreting more of the ER chaperone Kar2p, localizing Rer1p mostly to the vacuole, and increasing sensitivity to neomycin. Overexpression of ubiquitin in ret1-1 rsp5-1 mutant suppresses vacuolar accumulation of Rer1p. We found that the effect of rsp5 mutation on the Golgi-to-ER trafficking is similar to that of sla1Δ mutation in a gene encoding actin cytoskeleton proteins, an Rsp5p substrate. Additionally, Rsp5 and Sla1 proteins were found by co-immunoprecipitation in a complex containing COPI subunits. Together, our results show that Rsp5 ligase plays a role in regulating retrograde Golgi-to-ER trafficking.
COPI mediates retrograde trafficking from the Golgi to the endoplasmic reticulum (ER) and within the Golgi stack, sorting transmembrane proteins bearing C-terminal KKxx or KxKxx motifs. The structure of KxKxx motifs bound to the N-terminal WD-repeat domain of β'-COP identifies electrostatic contacts between the motif and complementary patches at the center of the β'-COP propeller. An absolute requirement of a two-residue spacing between the terminal carboxylate group and first lysine residue results from interactions of carbonyl groups in the motif backbone with basic side chains of β'-COP. Similar interactions are proposed to mediate binding of KKxx motifs by the homologous α-COP domain. Mutation of key interacting residues in either domain or in their cognate motifs abolishes in vitro binding and results in mistrafficking of dilysine-containing cargo in yeast without compromising cell viability. Flexibility between β'-COP WD-repeat domains and the location of cargo binding have implications for COPI coat assembly.
► Dilysine motifs bind the top surface of β'- and α-COP N-terminal WD-repeat domains ► Mutation of key binding site residues abolishes in vitro binding to dilysine motifs ► Loss of binding site prevents retrograde trafficking of dilysine motifs in vivo ► Mutants lacking dilysine motif binding site can transport other retrograde cargoes
COPI facilitates trafficking of transmembrane proteins bearing C-terminal KKxx or KxKxx motifs between the Golgi and the ER and within the Golgi stacks. Jackson et al. now provide molecular characterization of COPI cargo binding through elucidation of the structure of β'-COP N-terminal WD-repeat domain in complex with a KxKxx motif.
Coat protein complex I (COPI) vesicles, coated by seven coatomer subunits, are mainly responsible for Golgi-to-ER transport. Silkworm posterior silkgland (PSG), a highly differentiated secretory tissue, secretes fibroin for silk production, but many physiological processes in the PSG cells await further investigation.
Here, to investigate the role of silkworm COPI, we cloned six silkworm COPI subunits (α,β,β′, δ, ε, and ζ-COP), determined their peak expression in day 2 in fifth-instar PSG, and visualized the localization of COPI, as a coat complex, with cis-Golgi. By dsRNA injection into silkworm larvae, we suppressed the expression of α-, β′- and γ-COP, and demonstrated that COPI subunits were required for PSG tube expansion. Knockdown of α-COP disrupted the integrity of Golgi apparatus and led to a narrower glandular lumen of the PSG, suggesting that silkworm COPI is essential for PSG tube expansion.
The initial characterization reveals the essential roles of silkworm COPI in PSG. Although silkworm COPI resembles the previously characterized coatomers in other organisms, some surprising findings require further investigation. Therefore, our results suggest the silkworm as a model for studying intracellular transport, and would facilitate the establishment of silkworm PSG as an efficient bioreactor.
The GTPase Rab1 regulates endoplasmic reticulum-Golgi and early Golgi traffic. The guanine nucleotide exchange factor (GEF) or factors that activate Rab1 at these stages of the secretory pathway are currently unknown. Trs130p is a subunit of the yeast TRAPPII (transport protein particle II) complex, a multisubunit tethering complex that is a GEF for the Rab1 homologue Ypt1p. Here, we show that mammalian Trs130 (mTrs130) is a component of an analogous TRAPP complex in mammalian cells, and we describe for the first time the role that this complex plays in membrane traffic. mTRAPPII is enriched on COPI (Coat Protein I)-coated vesicles and buds, but not Golgi cisternae, and it specifically activates Rab1. In addition, we find that mTRAPPII binds to γ1COP, a COPI coat adaptor subunit. The depletion of mTrs130 by short hairpin RNA leads to an increase of vesicles in the vicinity of the Golgi and the accumulation of cargo in an early Golgi compartment. We propose that mTRAPPII is a Rab1 GEF that tethers COPI-coated vesicles to early Golgi membranes.
When transport between the rough endoplasmic reticulum (ER) and Golgi complex is blocked by Brefeldin A (BFA) treatment or ATP depletion, the Golgi apparatus and associated transport vesicles undergo a dramatic reorganization. Because recent studies suggest that coat proteins such as beta-COP play an important role in the maintenance of the Golgi complex, we have used immunocytochemistry to determine the distribution of beta-COP in pancreatic acinar cells (PAC) in which ER to Golgi transport was blocked by BFA treatment or ATP depletion. In controls, beta-COP was associated with Golgi cisternae and transport vesicles as expected. Upon BFA treatment, PAC Golgi cisternae are dismantled and replaced by clusters of remnant vesicles surrounded by typical ER transitional elements that are generally assumed to represent the exit site of vesicular carriers for ER to Golgi transport. In BFA-treated PAC, beta-COP was concentrated in large (0.5-1.0 micron) aggregates closely associated with remnant Golgi membranes. In addition to typical ER transitional elements, we detected a new type of transitional element that consists of specialized regions of rough ER (RER) with ribosome-free ends that touched or extended into the beta-COP containing aggregates. In ATP-depleted PAC, beta-COP was not detected on Golgi membranes but was concentrated in similar large aggregates found on the cis side of the Golgi stacks. The data indicate that upon arrest of ER to Golgi transport by either BFA treatment or energy depletion, beta-COP dissociates from PAC Golgi membranes and accumulates as large aggregates closely associated with specialized ER elements. The latter may correspond to either the site of entry or exit for vesicles recycling between the Golgi and the RER.
Endoplasmic reticulum (ER) α-1, 2-mannosidase and γ-COP contribute to a Golgi-based quality control module that facilitates the retrieval of captured ER-associated protein degradation substrates back to the ER.
Endoplasmic reticulum (ER) α-1, 2-mannosidase (ERManI) contributes to ER-associated protein degradation (ERAD) by initiating the formation of degradation signals on misfolded N-linked glycoproteins. Despite its inferred intracellular location, we recently discovered that the mammalian homologue is actually localized to the Golgi complex. In the present study, the functional role of Golgi-situated ERManI was investigated. Mass spectrometry analysis and coimmunoprecipitation (co-IP) identified a direct interaction between ERManI and γ-COP, the gamma subunit of coat protein complex I (COPI) that is responsible for Golgi-to-ER retrograde cargo transport. The functional relationship was validated by the requirement of both ERManI and γ-COP to support efficient intracellular clearance of the classical ERAD substrate, null Hong Kong (NHK). In addition, site-directed mutagenesis of suspected γ-COP–binding motifs in the cytoplasmic tail of ERManI was sufficient to disrupt the physical interaction and ablate NHK degradation. Moreover, a physical interaction between NHK, ERManI, and γ-COP was identified by co-IP and Western blotting. RNA interference–mediated knockdown of γ-COP enhanced the association between ERManI and NHK, while diminishing the efficiency of ERAD. Based on these findings, a model is proposed in which ERManI and γ-COP contribute to a Golgi-based quality control module that facilitates the retrieval of captured ERAD substrates back to the ER.
Simian virus 40 (SV40) enters cells by atypical endocytosis mediated by caveolae that transports the virus to the endoplasmic reticulum (ER) instead of to the endosomal-lysosomal compartment, which is the usual destination for viruses and other cargo that enter by endocytosis. We show here that SV4O is transported to the ER via an intermediate compartment that contains β-COP, which is best known as a component of the COPI coatamer complexes that are required for the retrograde retrieval pathway from the Golgi to the ER. Additionally, transport of SV40 to the ER, as well as infection, is sensitive to brefeldin A. This drug acts by specifically inhibiting the ARF1 GTPase, which is known to regulate assembly of COPI coat complexes on Golgi cisternae. Moreover, some β-COP colocalizes with intracellular caveolin-1, which was previously shown to be present on a new organelle (termed the caveosome) that is an intermediate in the transport of SV40 to the ER (L. Pelkmans, J. Kartenbeck, and A. Helenius, Nat. Cell Biol. 3:473-483, 2001). We also show that the internal SV40 capsid proteins VP2 and VP3 become accessible to immunostaining starting at about 5 h. Most of that immunostaining overlays the ER, with some appearing outside of the ER. In contrast, immunostaining with anti-SV40 antisera remains confined to the ER.
The genus Enterovirus, belonging to the family Picornaviridae, includes well-known pathogens, such as poliovirus, coxsackievirus, and rhinovirus. Brefeldin A (BFA) impedes replication of several enteroviruses through inhibition of Golgi-specific BFA resistance factor 1 (GBF1), a regulator of secretory pathway integrity and transport. GBF1 mediates the GTP exchange of Arf1, which in activated form recruits coatomer protein complex I (COP-I) to Golgi vesicles, a process important in transport between the endoplasmic reticulum and Golgi vesicles. Recently, the drugs AG1478 and Golgicide A (GCA) were put forward as new inhibitors of GBF1. In this study, we investigated the effects of these putative GBF1 inhibitors on secretory pathway function and enterovirus replication. We show that both drugs induced fragmentation of the Golgi vesicles and caused dissociation of Arf1 and COP-I from Golgi membranes, yet they differed in their effect on GBF1 localization. The effects of AG1478, but not those of GCA, could be countered by overexpression of Arf1, indicating a difference in their molecular mechanism of action. Consistent with this idea, we observed that GCA drastically reduced replication of coxsackievirus B3 (CVB3) and other human enterovirus species, whereas AG1478 had no effect at all on enterovirus replication. Time-of-addition studies and analysis of RNA replication using a subgenomic replicon both showed that GCA suppresses RNA replication of CVB3, which could be countered by overexpression of GBF1. These results indicate that, in contrast to AG1478, GCA inhibits CVB3 RNA replication by targeting GBF1. AG1478 and GCA may be valuable tools to further dissect enterovirus replication.
Coatomer is required for the retrieval of proteins from an early Golgi compartment back to the endoplasmic reticulum. The WD40 domain of α-COP is required for the recruitment of KKTN-tagged proteins into coatomer-coated vesicles. However, lack of the domain has only minor effects on growth in yeast. Here, we show that the WD40 domain of β′-COP is required for the recycling of the KTKLL-tagged Golgi protein Emp47p. The protein is degraded more rapidly in cells with a point mutation in the WD40 domain of β′-COP (sec27-95) or in cells lacking the domain altogether, whereas a point mutation in the Clathrin Heavy Chain Repeat (sec27-1) does not affect the turnover of Emp47p. Lack of the WD40 domain of β′-COP has only minor effects on growth of yeast cells; however, absence of both WD40 domains of α- and β′-COP is lethal. Two hybrid studies together with our analysis of the maturation of KKTN-tagged invertase and the turnover of Emp47p in α- and β′-COP mutants suggest that the two WD40 domains of α- and β′-COP bind distinct but overlapping sets of di-lysine signals and hence both contribute to recycling of proteins with di-lysine signals.
Coatomer is a cytosolic protein complex that forms the coat of COP I- coated transport vesicles. In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, [COPs] alpha-zeta), we engaged in a program to clone and characterize the individual coatomer subunits. We have now cloned, sequenced, and overexpressed bovine alpha-COP, the 135-kD subunit of coatomer as well as delta-COP, the 57-kD subunit and have identified a yeast homolog of delta-COP by cDNA sequence comparison and by NH2-terminal peptide sequencing. delta-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2. We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP I-coated transport vesicles and in the budding regions of the Golgi membranes. A knock-out of the delta-COP gene in yeast is lethal. Immunoprecipitation, as well as analysis exploiting the two-hybrid system in a complete COP screen, showed physical interactions between alpha- and epsilon-COPs and between beta- and delta-COPs. Moreover, the two-hybrid system indicates interactions between gamma- and zeta-COPs as well as between alpha- and beta' COPs. We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex.
The type I membrane protein calnexin is a conserved key component of the quality control mechanism in the endoplasmic reticulum. It functions as a molecular chaperone that monitors the folding state of nascent polypeptides entering the endoplasmic reticulum. Calnexin also behaves as a lectin, as its chaperoning activity involves binding of oligosaccharide moieties present on newly imported glycoproteins. We isolated the calnexin gene (HpCNE1) from the methylotrophic yeast Hansenula polymorpha, and used HpCNE1 expression plasmids for supertransformation of H. polymorpha strains secreting target proteins of biotechnological interest. The elevated dosage of HpCNE1 enhanced secretion of the four proteins tested: three glycoproteins and one unglycosylated product. Secretion of bacterial alginate epimerase AlgE1 was increased threefold on average, and secretion of both human interferon-γ and fungal consensus phytase twofold. With phytase and AlgE1 this improvement was all the more remarkable, as the secretion level was already high in the original strains (g L−1 range). The same approach improved secretion of human serum albumin, which lacks N-linked glycans, about twofold. Glycosylation of the pro-MFα1 leader may account for the effect of calnexin in this case. Our results argue that cooverexpression of calnexin can serve as a generally applicable tool for enhancing the secretion of all types of heterologous protein by H. polymorpha.
calnexin; Hansenula polymorpha; chaperone; heterologous protein expression; secretion
Picornavirus RNA replication requires the formation of replication complexes (RCs) consisting of virus-induced vesicles associated with viral nonstructural proteins and RNA. Brefeldin A (BFA) has been shown to strongly inhibit RNA replication of poliovirus but not of encephalomyocarditis virus (EMCV). Here, we demonstrate that the replication of parechovirus 1 (ParV1) is partly resistant to BFA, whereas echovirus 11 (EV11) replication is strongly inhibited. Since BFA inhibits COPI-dependent steps in endoplasmic reticulum (ER)-Golgi transport, we tested a hypothesis that different picornaviruses may have differential requirements for COPI in the formation of their RCs. Using immunofluorescence and cryo-immunoelectron microscopy we examined the association of a COPI component, β-COP, with the RCs of EMCV, ParV1, and EV11. EMCV RCs did not contain β-COP. In contrast, β-COP appeared to be specifically distributed to the RCs of EV11. In ParV1-infected cells β-COP was largely dispersed throughout the cytoplasm, with some being present in the RCs. These results suggest that there are differences in the involvement of COPI in the formation of the RCs of various picornaviruses, corresponding to their differential sensitivity to BFA. EMCV RCs are likely to be formed immediately after vesicle budding from the ER, prior to COPI association with membranes. ParV1 RCs are formed from COPI-containing membranes but COPI is unlikely to be directly involved in their formation, whereas formation of EV11 RCs appears to be dependent on COPI association with membranes.
COPI, a protein complex consisting of coatomer and the small GTPase
ARF1, is an integral component of some intracellular transport
carriers. The association of COPI with secretory membranes has been
implicated in the maintenance of Golgi integrity and the normal
functioning of intracellular transport in eukaryotes. The regulator of
G protein signaling, RGS4, interacted with the COPI subunit β′-COP in
a yeast two-hybrid screen. Both recombinant RGS4 and RGS2 bound
purified recombinant β′-COP in vitro. Endogenous cytosolic RGS4 from
NG108 cells and RGS2 from HEK293T cells cofractionated with the
COPI complex by gel filtration. Binding of β′-COP to RGS4 occurred
through two dilysine motifs in RGS4, similar to those contained in some
aminoglycoside antibiotics that are known to bind coatomer. RGS4
inhibited COPI binding to Golgi membranes independently of its
GTPase-accelerating activity on Giα. In RGS4-transfected
LLC-PK1 cells, the amount of COPI in the Golgi region was considerably
reduced compared with that in wild-type cells, but there was no
detectable difference in the amount of either Golgi-associated ARF1 or
the integral Golgi membrane protein giantin, indicating that Golgi
integrity was preserved. In addition, RGS4 expression inhibited
trafficking of aquaporin 1 to the plasma membrane in LLC-PK1 cells and
impaired secretion of placental alkaline phosphatase from HEK293T
cells. The inhibitory effect of RGS4 in these assays was independent of
GTPase-accelerating activity but correlated with its ability to bind
COPI. Thus, these data support the hypothesis that these RGS proteins
sequester coatomer in the cytoplasm and inhibit its recruitment onto
Golgi membranes, which may in turn modulate Golgi–plasma membrane or
What is the first membrane fusion step in the secretory pathway? In mammals, transport vesicles coated with coat complex (COP) II deliver secretory cargo to vesicular tubular clusters (VTCs) that ferry cargo from endoplasmic reticulum exit sites to the Golgi stack. However, the precise origin of VTCs and the membrane fusion step(s) involved have remained experimentally intractable. Here, we document in vitro direct tethering and SNARE-dependent fusion of endoplasmic reticulum–derived COPII transport vesicles to form larger cargo containers. The assembly did not require detectable Golgi membranes, preexisting VTCs, or COPI function. Therefore, COPII vesicles appear to contain all of the machinery to initiate VTC biogenesis via homotypic fusion. However, COPI function enhanced VTC assembly, and early VTCs acquired specific Golgi components by heterotypic fusion with Golgi-derived COPI vesicles.
Physiological conditions that impinge on constitutive traffic and affect organelle structure are not known. We report that osmotically induced cell volume changes, which are known to occur under a variety of conditions, rapidly inhibited endoplasmic reticulum (ER)-to-Golgi transport in mammalian cells. Both ER export and ER Golgi intermediate compartment (ERGIC)-to-Golgi trafficking steps were blocked, but retrograde transport was active, and it mediated ERGIC and Golgi collapse into the ER. Extensive tubulation and relatively rapid Golgi resident redistribution were observed under hypo-osmotic conditions, whereas a slower redistribution of the same markers, without apparent tubulation, was observed under hyperosmotic conditions. The osmotic stress response correlated with the perturbation of COPI function, because both hypo- and hyperosmotic conditions slowed brefeldin A-induced dissociation of βCOP from Golgi membranes. Remarkably, Golgi residents reemerged after several hours of sustained incubation in hypotonic or hypertonic medium. Reemergence was independent of new protein synthesis but required PKC, an activity known to mediate cell volume recovery. Taken together these results indicate the existence of a coupling between cell volume and constitutive traffic that impacts organelle structure through independent effects on anterograde and retrograde flow and that involves, in part, modulation of COPI function.
Membrane trafficking is essential to eukaryotic life and is controlled by a complex network of proteins that regulate movement of proteins and lipids between organelles. The GBF1/GEA family of Guanine nucleotide Exchange Factors (GEFs) regulates trafficking between the endoplasmic reticulum and Golgi by catalyzing the exchange of GDP for GTP on ADP Ribosylation Factors (Arfs). Activated Arfs recruit coat protein complex 1 (COP-I) to form vesicles that ferry cargo between these organelles. To further explore the function of the GBF1/GEA family, we have characterized a fission yeast mutant lacking one copy of the essential gene gea1 (gea1+/−), the Schizosaccharomyces pombe ortholog of GBF1. The haploinsufficient gea1+/− strain was shown to be sensitive to the GBF1 inhibitor brefeldin A (BFA) and was rescued from BFA sensitivity by gea1p overexpression. No overt defects in localization of arf1p or arf6p were observed in gea1+/− cells, but the fission yeast homolog of the COP-I cargo sac1 was mislocalized, consistent with impaired COP-I trafficking. Although Golgi morphology appeared normal, a slight increase in vacuolar size was observed in the gea1+/− mutant strain. Importantly, gea1+/− cells exhibited dramatic cytokinesis-related defects, including disorganized contractile rings, an increased septation index, and alterations in septum morphology. Septation defects appear to result from altered secretion of enzymes required for septum dynamics, as decreased secretion of eng1p, a β-glucanase required for septum breakdown, was observed in gea1+/− cells, and overexpression of eng1p suppressed the increased septation phenotype. These observations implicate gea1 in regulation of septum breakdown and establish S. pombe as a model system to explore GBF1/GEA function in cytokinesis.
Toxoplasma gondii and its apicomplexan relatives (such as Plasmodium falciparum, which causes malaria) are obligate intracellular parasites that rely on sequential protein release from specialized secretory organelles for invasion and multiplication within host cells. Because of the importance of these unusual membrane trafficking pathways for drug development and comparative cell biology, characterizing them is essential. In particular, it is unclear what role retrieval mechanisms play in parasite membrane trafficking or where they operate. Previously, we showed that T. gondii’s beta-COP (TgΒCOP; a subunit of coatomer protein complex I, COPI) and retrieval reporters localize exclusively to the zone between the parasite endoplasmic reticulum (ER) and Golgi apparatus. This suggested the existence of an HDEL receptor in T. gondii. We have now identified, cloned, and sequenced this receptor, TgERD2. TgERD2 localizes in a Golgi or ER pattern suggestive of the HDEL retrieval reporter (K. M. Hager, B. Striepen, L. G. Tilney, and D. S. Roos, J. Cell Sci. 112:2631-2638, 1999). A functional assay reveals that TgERD2 is able to complement the Saccharomyces cerevisiae ERD2 null mutant. Retrieval studies reveal that stable expression of a fluorescent exogenous retrieval ligand results in a dispersal of βCOP signal throughout the cytoplasm and, surprisingly, results in βCOP staining of the vacuolar space of the parasite. In contrast, stable expression of TgERD2GFP does not appear to disturb βCOP staining. In addition to TgERD2, Toxoplasma contains two more divergent ERD2 relatives. Phylogenetic analysis reveals that these proteins belong to a previously unrecognized ERD2 subfamily common to plants and alveolate organisms and as such could represent mediators of parasite-specific retrieval functions. No evidence of class 2 ERD2 proteins was found in metazoan organisms or fungi.
The 3A protein of the coxsackievirus B3 (CVB3), an enterovirus that belongs to the family of the picornaviruses, inhibits endoplasmic reticulum-to-Golgi transport. Recently, we elucidated the underlying mechanism by showing that CVB3 3A interferes with ADP-ribosylation factor 1 (Arf1)-dependent COP-I recruitment to membranes by binding and inhibiting the function of GBF1, a guanine nucleotide exchange factor that is required for the activation of Arf1 (E. Wessels et al., Dev. Cell 11:191-201, 2006). Here, we show that the 3A protein of poliovirus, another enterovirus, is also able to interfere with COP-I recruitment through the same mechanism. No interference with protein transport or COP-I recruitment was observed for the 3A proteins of any of the other picornaviruses tested here (human rhinovirus [HRV], encephalomyocarditis virus, foot-and-mouth disease virus, and hepatitis A virus). We show that the 3A proteins of HRV, which are the most closely related to the enteroviruses, are unable to inhibit COP-I recruitment, due to a reduced ability to bind GBF1. When the N-terminal residues of the HRV 3A proteins are replaced by those of CVB3 3A, chimeric proteins are produced that have gained the ability to bind GBF1 and, by consequence, to inhibit protein transport. These results show that the N terminus of the CVB3 3A protein is important for binding of GBF1 and its transport-inhibiting function. Taken together, our data demonstrate that the activity of the enterovirus 3A protein to inhibit GBF1-dependent COP-I recruitment is unique among the picornaviruses.
The coatomer (COPI) complex mediates Golgi to ER recycling of membrane proteins containing a dilysine retrieval motif. However, COPI was initially characterized as an anterograde-acting coat complex. To investigate the direct and primary role(s) of COPI in ER/Golgi transport and in the secretory pathway in general, we used PCR-based mutagenesis to generate new temperature-conditional mutant alleles of one COPI gene in Saccharomyces cerevisiae, SEC21 (γ-COP). Unexpectedly, all of the new sec21 ts mutants exhibited striking, cargo-selective ER to Golgi transport defects. In these mutants, several proteins (i.e., CPY and α-factor) were completely blocked in the ER at nonpermissive temperature; however, other proteins (i.e., invertase and HSP150) in these and other COPI mutants were secreted normally. Nearly identical cargo-specific ER to Golgi transport defects were also induced by Brefeldin A. In contrast, all proteins tested required COPII (ER to Golgi coat complex), Sec18p (NSF), and Sec22p (v-SNARE) for ER to Golgi transport. Together, these data suggest that COPI plays a critical but indirect role in anterograde transport, perhaps by directing retrieval of transport factors required for packaging of certain cargo into ER to Golgi COPII vesicles. Interestingly, CPY–invertase hybrid proteins, like invertase but unlike CPY, escaped the sec21 ts mutant ER block, suggesting that packaging into COPII vesicles may be mediated by cis-acting sorting determinants in the cargo proteins themselves. These hybrid proteins were efficiently targeted to the vacuole, indicating that COPI is also not directly required for regulated Golgi to vacuole transport. Additionally, the sec21 mutants exhibited early Golgi-specific glycosylation defects and structural aberrations in early but not late Golgi compartments at nonpermissive temperature. Together, these studies demonstrate that although COPI plays an important and most likely direct role both in Golgi–ER retrieval and in maintenance/function of the cis-Golgi, COPI does not appear to be directly required for anterograde transport through the secretory pathway.
The small GTPase Rab1b is essential for endoplasmic reticulum (ER) to Golgi transport, but its exact function remains unclear. We have examined the effects of wild-type and three mutant forms of Rab1b in vivo. We show that the inactive form of Rab1b (the N121I mutant with impaired guanine nucleotide binding) blocks forward transport of cargo and induces Golgi disruption. The phenotype is analogous to that induced by brefeldin A (BFA): it causes resident Golgi proteins to relocate to the ER and induces redistribution of ER-Golgi intermediate compartment proteins to punctate structures. The COPII exit machinery seems to be functional in cells expressing the N121I mutant, but COPI is compromised, as shown by the release of β-COP into the cytosol. Our results suggest that Rab1b function influences COPI recruitment. In support of this, we show that the disruptive effects of N121I can be reversed by expressing known mediators of COPI recruitment, the GTPase ARF1 and its guanine nucleotide exchange factor GBF1. Further evidence is provided by the finding that cells expressing the active form of Rab1b (the Q67L mutant with impaired GTPase activity) are resistant to BFA. Our data suggest a novel role for Rab1b in ARF1- and GBF1-mediated COPI recruitment pathway.
When a growing cell expands, lipids and proteins must be delivered to its periphery. Although this phenomenon has been observed for decades, it remains unknown how the secretory pathway responds to growth signaling. We demonstrate that control of Golgi phosphatidylinositol-4-phosphate (PI(4)P) is required for growth-dependent secretion. The phosphoinositide phosphatase SAC1 accumulates at the Golgi in quiescent cells and down-regulates anterograde trafficking by depleting Golgi PI(4)P. Golgi localization requires oligomerization of SAC1 and recruitment of the coat protein (COP) II complex. When quiescent cells are stimulated by mitogens, SAC1 rapidly shuttles back to the endoplasmic reticulum (ER), thus releasing the brake on Golgi secretion. The p38 mitogen-activated kinase (MAPK) pathway induces dissociation of SAC1 oligomers after mitogen stimulation, which triggers COP-I–mediated retrieval of SAC1 to the ER. Inhibition of p38 MAPK abolishes growth factor–induced Golgi-to-ER shuttling of SAC1 and slows secretion. These results suggest direct roles for p38 MAPK and SAC1 in transmitting growth signals to the secretory machinery.
Rab2 immunolocalizes to pre-Golgi intermediates (vesicular-tubular clusters [VTCs]) that are the first site of segregation of anterograde- and retrograde-transported proteins and a major peripheral site for COPI recruitment. Our previous work showed that Rab2 Q65L (equivalent to Ras Q61L) inhibited endoplasmic reticulum (ER)-to-Golgi transport in vivo. In this study, the biochemical properties of Rab2 Q65L were analyzed. The mutant protein binds GDP and GTP and has a low GTP hydrolysis rate that suggests that Rab2 Q65L is predominantly in the GTP-bound–activated form. The purified protein arrests vesicular stomatitis virus glycoprotein transport from VTCs in an assay that reconstitutes ER-to-Golgi traffic. A quantitative binding assay was used to measure membrane binding of β-COP when incubated with the mutant. Unlike Rab2 that stimulates recruitment, Rab2 Q65L showed a dose-dependent decrease in membrane-associated β-COP when incubated with rapidly sedimenting membranes (ER, pre-Golgi, and Golgi). The mutant protein does not interfere with β-COP binding but stimulates the release of slowly sedimenting vesicles containing Rab2, β-COP, and p53/gp58 but lacking anterograde grade-directed cargo. To complement the biochemical results, we observed in a morphological assay that Rab2 Q65L caused vesiculation of VTCs that accumulated at 15°C. These data suggest that the Rab2 protein plays a role in the low-temperature–sensitive step that regulates membrane flow from VTCs to the Golgi complex and back to the ER.
Biochemical data have shown that COPI-coated vesicles are tethered to Golgi membranes by a complex of at least three proteins: p115, giantin, and GM130. p115 binds to giantin on the vesicles and to GM130 on the membrane. We now examine the function of this tethering complex in vivo. Microinjection of an N-terminal peptide of GM130 or overexpression of GM130 lacking this N-terminal peptide inhibits the binding of p115 to Golgi membranes. Electron microscopic analysis of single microinjected cells shows that the number of COP-sized transport vesicles in the Golgi region increases substantially, suggesting that transport vesicles continue to bud but are less able to fuse. This was corroborated by quantitative immunofluorescence analysis, which showed that the intracellular transport of the VSV-G protein was significantly inhibited. Together, these data suggest that this tethering complex increases the efficiency with which transport vesicles fuse with their target membrane. They also provide support for a model of mitotic Golgi fragmentation in which the tethering complex is disrupted by mitotic phosphorylation of GM130.