Multiple activities for Arf1 at the Golgi complex

The Arf family of GTPases regulates membrane traffic and organelle structure. At the Golgi complex, Arf proteins facilitate membrane recruitment of many cytoplasmic coat proteins to allow sorting of membrane proteins for transport, stimulate the activity of enzymes that modulate the lipid composition of the Golgi, and assemble a cytoskeletal scaffold on the Golgi. Arf1 is the Arf family member most closely studied for its function at the Golgi complex. A number of regulators that activate and inactivate Arf1 on the Golgi have been described that localize to different regions of the organelle. This spatial distribution of Arf regulators may facilitate the recruitment of the coat proteins and other Arf effectors to different regions of the Golgi complex.     

The Rab6 GTPase regulates recruitment of the dynactin complex to Golgi membranes

Dynactin is a multisubunit protein complex required for the activity of dynein in diverse intracellular motility processes, including membrane transport. Dynactin can bind to vesicles and liposomes containing acidic phospholipids, but general properties such as this are unlikely to explain the regulated recruitment of dynactin to specific sites on organelle membranes. Additional factors must therefore exist to control this process. Candidates for these factors are the Rab GTPases, which function in the tethering of vesicles to their target organelle prior to membrane fusion. In particular, Rab27a tethers melanosomes to the actin cytoskeleton. Other Rabs have been implicated in microtubule-dependent organelle motility; Rab7 controls lysosomal transport, and Rab6 is involved in microtubule-dependent transport pathways through the Golgi and from endosomes to the Golgi. We demonstrate that dynactin binds to Rab6 and shows a Rab6-dependent recruitment to Golgi membranes. Other Golgi Rabs do not bind to dynactin and are unable to support its recruitment to membranes. Rab6 therefore functions as a specificity or tethering factor controlling the recruitment of dynactin to membranes.     

Mutations in a highly conserved region of the Arf1p activator GEA2 block anterograde Golgi transport but not COPI recruitment to membranes

We have identified an important functional region of the yeast Arf1 activator Gea2p upstream of the catalytic Sec7 domain and characterized a set of temperature-sensitive (ts) mutants with amino acid substitutions in this region. These gea2-ts mutants block or slow transport of proteins traversing the secretory pathway at exit from the endoplasmic reticulum (ER) and the early Golgi, and accumulate both ER and early Golgi membranes. No defects in two types of retrograde trafficking/sorting assays were observed. We find that a substantial amount of COPI is associated with Golgi membranes in the gea2-ts mutants, even after prolonged incubation at the nonpermissive temperature. COPI in these mutants is released from Golgi membranes by brefeldin A, a drug that binds directly to Gea2p and blocks Arf1 activation. Our results demonstrate that COPI function in sorting of at least three retrograde cargo proteins within the Golgi is not perturbed in these mutants, but that forward transport is severely inhibited. Hence this region of Gea2p upstream of the Sec7 domain plays a role in anterograde transport that is independent of its role in recruiting COPI for retrograde transport, at least of a subset of Golgi-ER cargo.     

The yeast Arf GTPase-activating protein Age1 is regulated by phospholipase D for post-Golgi vesicular transport

Vesicular transport shuttles cargo among intracellular compartments. Several stages of vesicular transport are mediated by the small GTPase Arf, which is controlled in a cycle of GTP binding and hydrolysis by Arf guanine-nucleotide exchange factors and Arf GTPase-activating proteins (ArfGAPs), respectively. In budding yeast the Age2 + Gcs1 ArfGAP pair facilitates post-Golgi transport. We have found the AGE1 gene, encoding another ArfGAP, can in high gene-copy number alleviate the temperature sensitivity of cells carrying mutations affecting the Age2 + Gcs1 ArfGAP pair. Moreover, increased AGE1 gene dosage compensates for the complete absence of the otherwise essential Age2 + Gcs1 ArfGAP pair. Increased dosage of SFH2, encoding a phosphatidylinositol transfer protein, also allows cell growth in the absence of the Age2 + Gcs1 pair, but good growth in this situation requires Age1. The ability of Age1 to overcome the need for Age2 + Gcs1 depends on phospholipase D activity that regulates lipid composition. We show by direct assessment of Age1 ArfGAP activity that Age1 is regulated by lipid composition and can provide ArfGAP function for post-Golgi transport.     

A PLA1-2 punch regulates the Golgi complex

The mammalian Golgi complex, trans Golgi network (TGN) and ER-Golgi intermediate compartment (ERGIC) are comprised of membrane cisternae, coated vesicles and membrane tubules, all of which contribute to membrane trafficking and maintenance of their unique architectures. Recently, a new cast of players was discovered to regulate the Golgi and ERGIC: four unrelated cytoplasmic phospholipase A (PLA) enzymes, cPLA(2)α (GIVA cPLA(2)), PAFAH Ib (GVIII PLA(2)), iPLA(2)-β (GVIA-2 iPLA(2)) and iPLA(1)γ. These ubiquitously expressed enzymes regulate membrane trafficking from specific Golgi subcompartments, although there is evidence for some functional redundancy between PAFAH Ib and cPLA(2)α. Three of these enzymes, PAFAH Ib, cPLA(2)α and iPLA(2)-β, exert effects on Golgi structure and function by inducing the formation of membrane tubules. We review our current understanding of how PLA enzymes regulate Golgi and ERGIC morphology and function.     

Pkh1/2-dependent phosphorylation of Vps27 regulates ESCRT-I recruitment to endosomes

Multivesicular endosomes (MVBs) are major sorting platforms for membrane proteins and participate in plasma membrane protein turnover, vacuolar/lysosomal hydrolase delivery, and surface receptor signal attenuation. MVBs undergo unconventional inward budding, which results in the formation of intraluminal vesicles (ILVs). MVB cargo sorting and ILV formation are achieved by the concerted function of endosomal sorting complex required for transport (ESCRT)-0 to ESCRT-III. The ESCRT-0 subunit Vps27 is a key player in this pathway since it recruits the other complexes to endosomes. Here we show that the Pkh1/Phk2 kinases, two yeast orthologues of the 3-phosphoinositide–dependent kinase, phosphorylate directly Vps27 in vivo and in vitro. We identify the phosphorylation site as the serine 613 and demonstrate that this phosphorylation is required for proper Vps27 function. Indeed, in pkh-ts temperature-sensitive mutant cells and in cells expressing vps27^S613A, MVB sorting of the carboxypeptidase Cps1 and of the α-factor receptor Ste2 is affected and the Vps28–green fluorescent protein ESCRT-I subunit is mainly cytoplasmic. We propose that Vps27 phosphorylation by Pkh1/2 kinases regulates the coordinated cascade of ESCRT complex recruitment at the endosomal membrane.     

Retrograde transport of the mannosyltransferase Och1p to the early Golgi requires a component of the COG transport complex

The yeast COG complex has been proposed to function as a vesicle-tethering complex on an early Golgi compartment, but its role is not fully understood. COG complex mutants exhibit a dramatic reduction in Golgi-specific glycosylation and other defects. Here we show that a strain carrying a COG3 temperature-sensitive allele, cog3-202, clearly exhibited the glycosylation defect while exhibiting nearly normal secretion kinetics. Two Golgi mannosyltransferases, Och1p and Mnn1p, were mislocalized in cog3-202 cells. In cog3-202 cells Och1-HA was found in lighter density membranes than in wild type cells. In sed5(ts) and sft1(ts) strains, Och1p rapidly accumulated in vesicle-like structures consistent with the delivery of Och1p back to the cis-Golgi on retrograde vesicles via a Sed5p/Sft1p-containing SNARE complex. In contrast to cog3-202 cells, the membranes in sed5(ts) cells that contained Och1p were denser than in wild type. Together these results indicate that Och1p does not accumulate in retrograde vesicles in the cog3-202 mutant and are consistent with the COG complex playing a role in sorting of Och1p into retrograde vesicles. In wild type cells Och1p has been shown previously to cycle between the cis-Golgi and minimally as far as the late Golgi. We find that Och1p does not cycle via endosomes during its normal itinerary suggesting that Och1p engages in intra-Golgi cycling only. However, Och1p does use a post-Golgi pathway for degradation because a portion of Och1p was degraded in the vacuole. Most surprisingly, Och1p can use either the carboxypeptidase Y or AP-3 pathways to reach the vacuole for degradation.     

CD99 regulates the transport of MHC class I molecules from the Golgi complex to the cell surface

The down-regulation of surface expression of MHC class I molecules has recently been reported in the CD99-deficient lymphoblastoid B cell line displaying the characteristics of Hodgkin's and Reed-Sternberg phenotype. Here, we demonstrate that the reduction of MHC class I molecules on the cell surface is primarily due to a defect in the transport from the Golgi complex to the plasma membrane. Loss of CD99 did not affect the steady-state expression levels of mRNA and protein of MHC class I molecules. In addition, the assembly of MHC class I molecules and the transport from the endoplasmic reticulum to the cis-Golgi occurred normally in the CD99-deficient cells, and no difference was detected between the CD99-deficient and the control cells in the pattern and degree of endocytosis. Instead, the CD99-deficient cells displayed the delayed transport of newly synthesized MHC class I molecules to the plasma membrane, thus causing accumulation of the molecules within the cells. The accumulated MHC class I molecules in the CD99-deficient cells were colocalized with alpha-mannosidase II and gamma-adaptin in the Golgi compartment. These results suggest that CD99 may be associated with the post-Golgi trafficking machinery by regulating the transport to the plasma membrane rather than the endocytosis of surface MHC class I molecules, providing a novel mechanism of MHC class I down-regulation for immune escape.     

ARAP1 regulates the ring size of circular dorsal ruffles through Arf1 and Arf5

Small guanosine triphosphatase (GTPase) ADP-ribosylation factors (Arfs) regulate membrane traffic and actin reorganization under the strict control of GTPase-activating proteins (GAPs). ARAP1 (Arf GAP with Rho GAP domain, ankyrin repeat, and PH domain 1) is an Arf GAP molecule with multiple PH domains that recognize phosphatidylinositol 3,4,5-trisphosphate. We found that growth factor stimulation induced localization of ARAP1 to an area of the plasma membrane inside the ring structure of circular dorsal ruffles (CDRs). Moreover, expression of ARAP1 increased the size of the CDR filamentous-actin ring in an Arf GAP activity-dependent manner, whereas smaller CDRs were formed by ARAP1 knockdown. In addition, expression of a dominant-negative mutant of Arf1 and Arf5, the substrates of ARAP1, expanded the size of CDRs, suggesting that the two Arf isoforms regulate ring structure downstream of ARAP1. Therefore our results reveal a novel molecular mechanism of CDR ring size control through the ARAP1-Arf1/5 pathway.     

Dynamics of GBF1, a Brefeldin A-sensitive Arf1 exchange factor at the Golgi

Trafficking through the Golgi apparatus requires members of the Arf family of GTPases, whose activation is regulated by guanine nucleotide exchange factors (GEFs). Once activated, Arf-GTP recruits effectors such as coat complexes and lipid-modifying enzymes to specific membrane sites, creating a domain competent for cargo concentration and transport. GBF1 is a peripherally associated Arf GEF involved in both endoplasmic reticulum-Golgi and intra-Golgi transport. The mechanism of GBF1 binding to membranes is unknown. As a first step to understanding the mechanism of membrane association, we constructed a yellow fluorescent protein-tagged version of GBF1 and performed fluorescence recovery after photobleaching analysis to determine its residence time on Golgi membranes. We find that GBF1 molecules are not stably associated with the Golgi but rather cycle rapidly on and off membranes. The drug brefeldin A (BFA), an uncompetitive inhibitor of the exchange reaction that binds to an Arf-GDP-Arf GEF complex, stabilizes GBF1 on Golgi membranes. Using an in vivo assay to monitor Arf1-GTP levels, we show that GBF1 exchange activity on Arf1 is inhibited by BFA in mammalian cells. These results suggest that an Arf1-GBF1-BFA complex is formed and has a longer residence time on Golgi membranes than GBF1 or Arf1 alone.     

Conserved oligomeric Golgi complex specifically regulates the maintenance of Golgi glycosylation machinery

Cell surface lectin staining, examination of Golgi glycosyltransferases stability and localization, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis were employed to investigate conserved oligomeric Golgi (COG)-dependent glycosylation defects in HeLa cells. Both Griffonia simplicifolia lectin-II and Galanthus nivalus lectins were specifically bound to the plasma membrane glycoconjugates of COG-depleted cells, indicating defects in activity of medial- and trans-Golgi-localized enzymes. In response to siRNA-induced depletion of COG complex subunits, several key components of Golgi glycosylation machinery, including MAN2A1, MGAT1, B4GALT1 and ST6GAL1, were severely mislocalized. MALDI-TOF analysis of total N-linked glycoconjugates indicated a decrease in the relative amount of sialylated glycans in both COG3 KD and COG4 KD cells. In agreement to a proposed role of the COG complex in retrograde membrane trafficking, all types of COG-depleted HeLa cells were deficient in the Brefeldin A- and Sar1 DN-induced redistribution of Golgi resident glycosyltransferases to the endoplasmic reticulum. The retrograde trafficking of medial- and trans-Golgi-localized glycosylation enzymes was affected to a larger extent, strongly indicating that the COG complex regulates the intra-Golgi protein movement. COG complex-deficient cells were not defective in Golgi re-assembly after the Brefeldin A washout, confirming specificity in the retrograde trafficking block. The lobe B COG subcomplex subunits COG6 and COG8 were localized on trafficking intermediates that carry Golgi glycosyltransferases, indicating that the COG complex is directly involved in trafficking and maintenance of Golgi glycosylation machinery.     

Rab1b regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments

We report an essential role for the ras-related small GTP-binding protein rab1b in vesicular transport in mammalian cells. mAbs detect rab1b in both the ER and Golgi compartments. Using an assay which reconstitutes transport between the ER and the cis-Golgi compartment, we find that rab1b is required during an initial step in export of protein from the ER. In addition, it is also required for transport of protein between successive cis- and medial-Golgi compartments. We suggest that rab1b may provide a common link between upstream and downstream components of the vesicular fission and fusion machinery functioning in early compartments of the secretory pathway.     

Src regulates Golgi structure and KDEL receptor-dependent retrograde transport to the endoplasmic reticulum

The tyrosine kinase Src is present on the Golgi membranes. Its role, however, in the overall function and organization of the Golgi apparatus is unclear. We have found that in a cell line called SYF, which lacks the three ubiquitous Src-like kinases (Src, Yes, and Fyn), the organization of the Golgi apparatus is perturbed. The Golgi apparatus is composed of collapsed stacks and bloated cisternae in these cells. Expression of an activated form of Src relocated the KDEL receptor (KDEL-R) from the Golgi apparatus to the endoplasmic reticulum. Other Golgi-specific marker proteins were not affected under these conditions. Because of the specific effect of Src on the location of KDEL-R, we tested whether protein transport between ER and the Golgi apparatus involves Src. Transport of Pseudomonas exotoxin, which is transported to the ER by binding to the KDEL-R is accelerated by inhibition or genetic ablation of Src. Protein transport from ER to the Golgi apparatus however, is unaffected by Src deletion or inhibition. We propose that Src has an appreciable role in the organization of the Golgi apparatus, which may be linked to its involvement in protein transport from the Golgi apparatus to the endoplasmic reticulum.     

GFP-golvesin constructs to study Golgi tubulation and post-Golgi vesicle dynamics in phagocytosis

Dictyostelium cells are professional phagocytes that are optimally suited for the imaging of phagosome processing from particle uptake to exocytosis. In order to design fluorescent probes for monitoring membrane trafficking in the endocytic pathway, we have dissected a membrane protein, golvesin, and have linked fragments of its sequence to GFP. Endogenous golvesin is partitioned between the ER, the Golgi apparatus, endosomes, and the contractile vacuole complex. We have localized signals that are required for exit from the Golgi to post-Golgi compartments to the C-terminal region of the golvesin sequence. One GFP-tagged fragment turned out to be a highly specific Golgi marker and was used to demonstrate the interaction of Golgi tubules with phagosomes. Signals essential for the retrieval of golvesin at the end of phagosome processing were localized to the N-terminal region. A truncated golvesin construct escaping retrieval was employed in recording the delivery of a phagosomal protein to the plasma membrane. Applying this construct to a phagosome filled with multiple particles, we observed that the phagosome is segmented during exocytosis, meaning that sequential release of particles alternates with membrane fusion.     

Evidence for a COP-I-independent transport route from the Golgi complex to the endoplasmic reticulum

The cytosolic coat-protein complex COP-I interacts with cytoplasmic 'retrieval' signals present in membrane proteins that cycle between the endoplasmic reticulum (ER) and the Golgi complex, and is required for both anterograde and retrograde transport in the secretory pathway. Here we study the role of COP-I in Golgi-to-ER transport of several distinct marker molecules. Microinjection of anti-COP-I antibodies inhibits retrieval of the lectin-like molecule ERGIC-53 and of the KDEL receptor from the Golgi to the ER. Transport to the ER of protein toxins, which contain a sequence that is recognized by the KDEL receptor, is also inhibited. In contrast, microinjection of anti-COP-I antibodies or expression of a GTP-restricted Arf-1 mutant does not interfere with Golgi-to-ER transport of Shiga toxin/Shiga-like toxin-1 or with the apparent recycling to the ER of Golgi-resident glycosylation enzymes. Overexpression of a GDP-restricted mutant of Rab6 blocks transport to the ER of Shiga toxin/Shiga-like toxin-1 and glycosylation enzymes, but not of ERGIC-53, the KDEL receptor or KDEL-containing toxins. These data indicate the existence of at least two distinct pathways for Golgi-to-ER transport, one COP-I dependent and the other COP-I independent. The COP-I-independent pathway is specifically regulated by Rab6 and is used by Golgi glycosylation enzymes and Shiga toxin/Shiga-like toxin-1.     

COPI-mediated retrograde trafficking from the Golgi to the ER regulates EGFR nuclear transport

Emerging evidence indicates that cell surface receptors, such as the entire epidermal growth factor receptor (EGFR) family, have been shown to localize in the nucleus. A retrograde route from the Golgi to the endoplasmic reticulum (ER) is postulated to be involved in the EGFR trafficking to the nucleus; however, the molecular mechanism in this proposed model remains unexplored. Here, we demonstrate that membrane-embedded vesicular trafficking is involved in the nuclear transport of EGFR. Confocal immunofluorescence reveals that in response to EGF, a portion of EGFR redistributes to the Golgi and the ER, where its NH₂-terminus resides within the lumen of Golgi/ER and COOH-terminus is exposed to the cytoplasm. Blockage of the Golgi-to-ER retrograde trafficking by brefeldin A or dominant mutants of the small GTPase ADP-ribosylation factor, which both resulted in the disassembly of the coat protein complex I (COPI) coat to the Golgi, inhibit EGFR transport to the ER and the nucleus. We further find that EGF-dependent nuclear transport of EGFR is regulated by retrograde trafficking from the Golgi to the ER involving an association of EGFR with γ-COP, one of the subunits of the COPI coatomer. Our findings experimentally provide a comprehensive pathway that nuclear transport of EGFR is regulated by COPI-mediated vesicular trafficking from the Golgi to the ER, and may serve as a general mechanism in regulating the nuclear transport of other cell surface receptors.     

Mon2, a relative of large Arf exchange factors, recruits Dop1 to the Golgi apparatus

The protein Mon2 is distantly related to the guanine nucleotide exchange factors (GEFs) that activate Arf1 on Golgi membranes. However, unlike these "large" Arf GEFs, Mon2 lacks the Sec7 domain that catalyzes nucleotide exchange on Arf1. Here we report that yeast Mon2 shares extensive homology with the noncatalytic parts of both the BIG and Golgi brefeldin A resistance factor subfamilies of Arf GEFs and is located to the trans-Golgi. Moreover, we find that Mon2 forms a complex with Dop1, a large cytoplasmic protein conserved in evolution from humans to protozoa. Deletion of Mon2 results in mislocalization of Dop1 from the Golgi and defects in cycling between endosomes and the Golgi. However, unlike Mon2, Dop1 is essential for yeast viability. A conditional allele of Dop1 shows that loss of Dop1 activity not only affects endosome to Golgi transport but also causes a severe perturbation of the organization of the endoplasmic reticulum. Thus, it appears that Dop1 plays a widespread role in membrane organization, and Mon2 acts as a scaffold to recruit the Golgi-localized pool of Dop1.     

Requirement for neo1p in retrograde transport from the Golgi complex to the endoplasmic reticulum

Neo1p from Saccharomyces cerevisiae is an essential P-type ATPase and potential aminophospholipid translocase (flippase) in the Drs2p family. We have previously implicated Drs2p in protein transport steps in the late secretory pathway requiring ADP-ribosylation factor (ARF) and clathrin. Here, we present evidence that epitope-tagged Neo1p localizes to the endoplasmic reticulum (ER) and Golgi complex and is required for a retrograde transport pathway between these organelles. Using conditional alleles of NEO1, we find that loss of Neo1p function causes cargo-specific defects in anterograde protein transport early in the secretory pathway and perturbs glycosylation in the Golgi complex. Rer1-GFP, a protein that cycles between the ER and Golgi complex in COPI and COPII vesicles, is mislocalized to the vacuole in neo1-ts at the nonpermissive temperature. These phenotypes suggest that the anterograde protein transport defect is a secondary consequence of a defect in a COPI-dependent retrograde pathway. We propose that loss of lipid asymmetry in the cis Golgi perturbs retrograde protein transport to the ER.