Reconstitution of COPII vesicle fusion to generate a pre-Golgi intermediate compartment

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.     

Isolation of Functional Golgi-derived Vesicles with a Possible Role in Retrograde Transport

Secretory proteins enter the Golgi apparatus when transport vesicles fuse with the cis-side and exit in transport vesicles budding from the trans-side. Resident Golgi enzymes that have been transported in the cis-to-trans direction with the secretory flow must be recycled constantly by retrograde transport in the opposite direction. In this study, we describe the functional characterization of Golgi-derived transport vesicles that were isolated from tissue culture cells. We found that under the steady-state conditions of a living cell, a fraction of resident Golgi enzymes was found in vesicles that could be separated from cisternal membranes. These vesicles appeared to be depleted of secretory cargo. They were capable of binding to and fusion with isolated Golgi membranes, and after fusion their enzymatic contents most efficiently processed cargo that had just entered the Golgi apparatus. Those results indicate a possible role for these structures in recycling of Golgi enzymes in the Golgi stack.     

ER to Golgi transport: Requirement for p115 at a pre-Golgi VTC stage

The membrane transport factor p115 functions in the secretory pathway of mammalian cells. Using biochemical and morphological approaches, we show that p115 participates in the assembly and maintenance of normal Golgi structure and is required for ER to Golgi traffic at a pre-Golgi stage. Injection of antibodies against p115 into intact WIF-B cells caused Golgi disruption and inhibited Golgi complex reassembly after BFA treatment and wash-out. Addition of anti-p115 antibodies or depletion of p115 from a VSVtsO45 based semi-intact cell transport assay inhibited transport. The inhibition occurred after VSV glycoprotein (VSV-G) exit from the ER but before its delivery to the Golgi complex, and resulted in VSV-G protein accumulating in peripheral vesicular tubular clusters (VTCs). The p115-requiring step of transport followed the rab1-requiring step and preceded the Ca(2+)-requiring step. Unexpectedly, mannosidase I redistributed from the Golgi complex to colocalize with VSV-G protein arrested in pre-Golgi VTCs by p115 depletion. Redistribution of mannosidase I was also observed in cells incubated at 15 degrees C. Our data show that p115 is essential for the translocation of pre-Golgi VTCs from peripheral sites to the Golgi stack. This defines a previously uncharacterized function for p115 at the VTC stage of ER to Golgi traffic.     

TFG clusters COPII‐coated transport carriers and promotes early secretory pathway organization

In mammalian cells, cargo‐laden secretory vesicles leave the endoplasmic reticulum (ER) en route to ER‐Golgi intermediate compartments (ERGIC) in a manner dependent on the COPII coat complex. We report here that COPII‐coated transport carriers traverse a submicron, TFG (Trk‐fused gene)‐enriched zone at the ER/ERGIC interface. The architecture of TFG complexes as determined by three‐dimensional electron microscopy reveals the formation of flexible, octameric cup‐like structures, which are able to self‐associate to generate larger polymers in vitro. In cells, loss of TFG function dramatically slows protein export from the ER and results in the accumulation of COPII‐coated carriers throughout the cytoplasm. Additionally, the tight association between ER and ERGIC membranes is lost in the absence of TFG. We propose that TFG functions at the ER/ERGIC interface to locally concentrate COPII‐coated transport carriers and link exit sites on the ER to ERGIC membranes. Our findings provide a new mechanism by which COPII‐coated carriers are retained near their site of formation to facilitate rapid fusion with neighboring ERGIC membranes upon uncoating, thereby promoting interorganellar cargo transport.     

Spatial partitioning of secretory cargo from Golgi resident proteins in live cells

Background  

To maintain organelle integrity, resident proteins must segregate from itinerant cargo during secretory transport. However, Golgi resident enzymes must have intimate access to secretory cargo in order to carry out glycosylation reactions. The amount of cargo and associated membrane may be significant compared to the amount of Golgi membrane and resident protein, but upon Golgi exit, cargo and resident are efficiently sorted. How this occurs in live cells is not known.     

The p24 family and selective transport processes at the ER—Golgi interface

The secretory pathway is of vital importance for eukaryotic cells and has a pivotal role in the synthesis, sorting, processing and secretion of a large variety of bioactive molecules involved in intercellular communication. One of the key processes in the secretory pathway concerns the transport of cargo proteins from the ER (endoplasmic reticulum) to the Golgi. Type‐I transmembrane proteins of ～24 kDa are abundantly present in the membranes of the early secretory pathway, and bind the COPI and COPII coat complexes that cover vesicles travelling between the membranes. These p24 proteins are thought to play an important role in the selective transport processes at the ER—Golgi interface, although their exact functioning is still obscure. One model proposes that p24 proteins couple cargo selection in the lumen with vesicle coat recruitment in the cytosol. Alternatively, p24 proteins may furnish subcompartments of the secretory pathway with the correct subsets of machinery proteins. Here we review the current knowledge of the p24 proteins and the various roles proposed for the p24 family members.     

Sequential coupling between COPII and COPI vesicle coats in endoplasmic reticulum to Golgi transport

COPI and COPII are vesicle coat complexes whose assembly is regulated by the ARF1 and Sar1 GTPases, respectively. We show that COPI and COPII coat complexes are recruited separately and independently to ER (COPII), pre-Golgi (COPI, COPII), and Golgi (COPI) membranes of mammalian cells. To address their individual roles in ER to Golgi transport, we used stage specific in vitro transport assays to synchronize movement of cargo to and from pre-Golgi intermediates, and GDP- and GTP-restricted forms of Sar1 and ARF1 proteins to control coat recruitment. We find that COPII is solely responsible for export from the ER, is lost rapidly following vesicle budding and mediates a vesicular step required for the build-up of pre-Golgi intermediates composed of clusters of vesicles and small tubular elements. COPI is recruited onto pre-Golgi intermediates where it initiates segregation of the anterograde transported protein vesicular stomatitis virus glycoprotein (VSV-G) from the retrograde transported protein p58, a protein which actively recycles between the ER and pre-Golgi intermediates. We propose that sequential coupling between COPII and COPI coats is essential to coordinate and direct bi-directional vesicular traffic between the ER and pre-Golgi intermediates involved in transport of protein to the Golgi complex.     

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.     

Grab a Golgi: Laser Trapping of Golgi Bodies Reveals in vivo Interactions with the Endoplasmic Reticulum

In many vacuolate plant cells, individual Golgi bodies appear to be attached to tubules of the pleiomorphic cortical endoplasmic reticulum (ER) network. Such observations culminated in the controversial mobile secretory unit hypothesis to explain transport of cargo from the ER to Golgi via Golgi attached export sites. This proposes that individual Golgi bodies and an attached‐ER exit machinery move over or with the surface of the ER whilst collecting cargo for secretion. By the application of infrared laser optical traps to individual Golgi bodies within living leaf cells, we show that individual Golgi bodies can be micromanipulated to reveal their association with the ER. Golgi bodies are physically attached to ER tubules and lateral displacement of individual Golgi bodies results in the rapid growth of the attached ER tubule. Remarkably, the ER network can be remodelled in living cells simply by movement of laser trapped Golgi dragging new ER tubules through the cytoplasm and new ER anchor sites can be established. Finally, we show that trapped Golgi ripped off the ER are ‘sticky’ and can be docked on to and attached to ER tubules, which will again show rapid growth whilst pulled by moving Golgi.     

Guanine nucleotide dissociation inhibitor is essential for Rab1 function in budding from the endoplasmic reticulum and transport through the Golgi stack

The small GTPase Rab1 is required for vesicular traffic from the ER to the cis-Golgi compartment, and for transport between the cis and medial compartments of the Golgi stack. In the present study, we examine the role of guanine nucleotide dissociation inhibitor (GDI) in regulating the function of Rab1 in the transport of vesicular stomatitis virus glycoprotein (VSV-G) in vitro. Incubation in the presence of excess GDI rapidly (t1/2 < 30 s) extracted Rab1 from membranes, inhibiting vesicle budding from the ER and sequential transport between the cis-, medial-, and trans-Golgi cisternae. These results demonstrate a direct role for GDI in the recycling of Rab proteins. Analysis of rat liver cytosol by gel filtration revealed that a major pool of Rab1 fractionates with a molecular mass of approximately 80 kD in the form of a GDI-Rab1 complex. When the GDI-Rab1 complex was depleted from cytosol by use of a Rab1-specific antibody, VSV-G failed to exit the ER. However, supplementation of depleted cytosol with a GDI-Rab1 complex prepared in vitro from recombinant forms of Rab1 and GDI efficiently restored export from the ER, and transport through the Golgi stack. These results provide evidence that a cytosolic GDI-Rab1 complex is required for the formation of non-clathrin-coated vesicles mediating transport through the secretory pathway.     

A positive signal prevents secretory membrane cargo from recycling between the Golgi and the ER

The Golgi complex and ER are dynamically connected by anterograde and retrograde trafficking pathways. To what extent and by what mechanism outward‐bound cargo proteins escape retrograde trafficking has been poorly investigated. Here, we analysed the behaviour of several membrane proteins at the ER/Golgi interface in live cells. When Golgi‐to‐plasma membrane transport was blocked, vesicular stomatitis virus glycoprotein (VSVG), which bears an ER export signal, accumulated in the Golgi, whereas an export signal‐deleted version of VSVG attained a steady state determined by the balance of retrograde and anterograde traffic. A similar behaviour was displayed by EGF receptor and by a model tail‐anchored protein, whose retrograde traffic was slowed by addition of VSVG's export signal. Retrograde trafficking was energy‐ and Rab6‐dependent, and Rab6 inhibition accelerated signal‐deleted VSVG's transport to the cell surface. Our results extend the dynamic bi‐directional relationship between the Golgi and ER to include surface‐directed proteins, uncover an unanticipated role for export signals at the Golgi complex, and identify recycling as a novel factor that regulates cargo transport out of the early secretory pathway.     

Quantitative ER <--> Golgi transport kinetics and protein separation upon Golgi exit revealed by vesicular integral membrane protein 36 dynamics in live cells

To quantitatively investigate the trafficking of the transmembrane lectin VIP36 and its relation to cargo-containing transport carriers (TCs), we analyzed a C-terminal fluorescent-protein (FP) fusion, VIP36-SP-FP. When expressed at moderate levels, VIP36-SP-FP localized to the endoplasmic reticulum, Golgi apparatus, and intermediate transport structures, and colocalized with epitope-tagged VIP36. Temperature shift and pharmacological experiments indicated VIP36-SP-FP recycled in the early secretory pathway, exhibiting trafficking representative of a class of transmembrane cargo receptors, including the closely related lectin ERGIC53. VIP36-SP-FP trafficking structures comprised tubules and globular elements, which translocated in a saltatory manner. Simultaneous visualization of anterograde secretory cargo and VIP36-SP-FP indicated that the globular structures were pre-Golgi carriers, and that VIP36-SP-FP segregated from cargo within the Golgi and was not included in post-Golgi TCs. Organelle-specific bleach experiments directly measured the exchange of VIP36-SP-FP between the Golgi and endoplasmic reticulum (ER). Fitting a two-compartment model to the recovery data predicted first order rate constants of 1.22 +/- 0.44%/min for ER --> Golgi, and 7.68 +/- 1.94%/min for Golgi --> ER transport, revealing a half-time of 113 +/- 70 min for leaving the ER and 1.67 +/- 0.45 min for leaving the Golgi, and accounting for the measured steady-state distribution of VIP36-SP-FP (13% Golgi/87% ER). Perturbing transport with AlF(4)(-) treatment altered VIP36-SP-GFP distribution and changed the rate constants. The parameters of the model suggest that relatively small differences in the first order rate constants, perhaps manifested in subtle differences in the tendency to enter distinct TCs, result in large differences in the steady-state localization of secretory components.     

Rab6 coordinates a novel Golgi to ER retrograde transport pathway in live cells

We visualized a fluorescent-protein (FP) fusion to Rab6, a Golgi-associated GTPase, in conjunction with fluorescent secretory pathway markers. FP-Rab6 defined highly dynamic transport carriers (TCs) translocating from the Golgi to the cell periphery. FP-Rab6 TCs specifically accumulated a retrograde cargo, the wild-type Shiga toxin B-fragment (STB), during STB transport from the Golgi to the endoplasmic reticulum (ER). FP-Rab6 TCs associated intimately with the ER, and STB entered the ER via specialized peripheral regions that accumulated FP-Rab6. Microinjection of antibodies that block coatomer protein I (COPI) function inhibited trafficking of a KDEL-receptor FP-fusion, but not FP-Rab6. Additionally, markers of COPI-dependent recycling were excluded from FP-Rab6/STB TCs. Overexpression of Rab6:GDP (T27N mutant) using T7 vaccinia inhibited toxicity of Shiga holotoxin, but did not alter STB transport to the Golgi or Golgi morphology. Taken together, our results indicate Rab6 regulates a novel Golgi to ER transport pathway.     

Release from Endoplasmic Reticulum Matrix Proteins Controls Cell Surface Transport of MHC Class I Molecules

The anterograde transport of secretory proteins from the endoplasmic reticulum (ER) to the plasma membrane is a multi‐step process. Secretory proteins differ greatly in their transport rates to the cell surface, but the contribution of each individual step to this difference is poorly understood. Transport rates may be determined by protein folding, chaperone association in the ER, access to ER exit sites (ERES) and retrieval from the ER‐Golgi intermediate compartment or the cis‐Golgi to the ER. We have used a combination of folding and trafficking assays to identify the differential step in the cell surface transport of two natural allotypes of the murine major histocompatibility complex (MHC) class I peptide receptor, H‐2D^b and H‐2K^b. We find that a novel pre‐ER exit process that acts on the folded lumenal part of MHC class I molecules and that drastically limits their access to ERES accounts for the transport difference of the two allotypes. Our observations support a model in which the cell surface transport of MHC class I molecules and other type I transmembrane proteins is governed by the affinity of all their folding and maturation states to the proteins of the ER matrix.      

Identification of ERGIC-53 as an intracellular transport receptor of alpha1-antitrypsin

Secretory proteins are exported from the endoplasmic reticulum (ER) by bulk flow and/or receptor-mediated transport. Our understanding of this process is limited because of the low number of identified transport receptors and cognate cargo proteins. In mammalian cells, the lectin ER Golgi intermediate compartment 53-kD protein (ERGIC-53) represents the best characterized cargo receptor. It assists ER export of a subset of glycoproteins including coagulation factors V and VIII and cathepsin C and Z. Here, we report a novel screening strategy to identify protein interactions in the lumen of the secretory pathway using a yellow fluorescent protein-based protein fragment complementation assay. By screening a human liver complementary DNA library, we identify alpha1-antitrypsin (alpha1-AT) as previously unrecognized cargo of ERGIC-53 and show that cargo capture is carbohydrate- and conformation-dependent. ERGIC-53 knockdown and knockout cells display a specific secretion defect of alpha1-AT that is corrected by reintroducing ERGIC-53. The results reveal ERGIC-53 to be an intracellular transport receptor of alpha1-AT and provide direct evidence for active receptor-mediated ER export of a soluble secretory protein in higher eukaryotes.     

Lectins and traffic in the secretory pathway

Evidence is accumulating that intracellular animal lectins play important roles in quality control and glycoprotein sorting along the secretory pathway. Calnexin and calreticulin in conjunction with associated chaperones promote correct folding and oligomerization of many glycoproteins in the endoplasmic reticulum (ER). The mannose lectin ERGIC-53 operates as a cargo receptor in transport of glycoproteins from ER to Golgi and the homologous lectin VIP36 may operate in quality control of glycosylation in the Golgi. Exit from the Golgi of lysosomal hydrolases to endosomes requires mannose 6-phosphate receptors and exit to the apical plasma membrane may also involve traffic lectins. Here we discuss the features of these lectins and their role in glycoprotein traffic in the secretory pathway.     

Sequential intermediates in the transport of protein between the endoplasmic reticulum and the Golgi

Semi-intact cells, a cell population in which the plasma membrane is perforated to expose intact intracellular organelles (Beckers, C. J. M., Keller, D. S., and Balch, W. E. (1987) Cell 50, 523-534), efficiently reconstitute vesicular trafficking of protein from the endoplasmic reticulum (ER) to the cis Golgi compartment. We now extend these studies to biochemically dissect transport of protein between the ER and the Golgi into a series of sequential intermediate steps involved in the budding and fusion of carrier vesicles. At least two broad categories of transport intermediates can be detected, those that involve early steps in transport and those involved in late, fusion-related events. Early transport steps require the transport of protein through a novel intermediate compartment in which protein accumulates at reduced temperature (15 degrees C). We demonstrate that both entry and exit from this 15 degrees C compartment can be successfully reconstituted in vitro. A late step in delivery of protein to the cis Golgi compartment requires Ca2+ (pCa7) and is coincident with a step which is sensitive to a peptide analog which blocks interaction between the Rab family of small GTP-binding proteins and a downstream effector protein(s) (Plutner, H., Schwaninger, R., Pind, S., and Balch, W. E. (1990) EMBO J. 9, 2375-2384). The combined results suggest that a single round of vesicular transport between the ER and the Golgi involves a rapid transit through N-ethylmaleimide-sensitive, guanosine 5'-(3-O-thio)triphosphate-sensitive, ATP- and cytosol-dependent step(s) involved in vesicle formation or transport to a novel intermediate compartment, followed by a regulated fusion event triggered in the presence of Ca2+ and functional components interacting with member(s) of the Rab gene family.     

The role of microtubules in transport between the endoplasmic reticulum and Golgi apparatus in mammalian cells

The organization of intracellular compartments and the transfer of components between them are central to the correct functioning of mammalian cells. Proteins and lipids are transferred between compartments by the formation, movement and subsequent specific fusion of transport intermediates. These vesicles and membrane clusters must be coupled to the cytoskeleton and to motor proteins that drive motility. Anterograde ER (endoplasmic reticulum)-to-Golgi transport, and the converse step of retrograde traffic from the Golgi to the ER, are now known to involve coupling of membranes to the microtubule cytoskeleton. Here we shall discuss our current understanding of the mechanisms that link membrane traffic in the early secretory pathway to the microtubule cytoskeleton in mammalian cells. Recent data have also provided molecular detail of functional co-ordination of motor proteins to specify directionality, as well as mechanisms for regulating motor activity by protein phosphorylation.     

Golgi matrix proteins interact with p24 cargo receptors and aid their efficient retention in the Golgi apparatus.

The Golgi apparatus is a highly complex organelle comprised of a stack of cisternal membranes on the secretory pathway from the ER to the cell surface. This structure is maintained by an exoskeleton or Golgi matrix constructed from a family of coiled-coil proteins, the golgins, and other peripheral membrane components such as GRASP55 and GRASP65. Here we find that TMP21, p24a, and gp25L, members of the p24 cargo receptor family, are present in complexes with GRASP55 and GRASP65 in vivo. GRASPs interact directly with the cytoplasmic domains of specific p24 cargo receptors depending on their oligomeric state, and mutation of the GRASP binding site in the cytoplasmic tail of one of these, p24a, results in it being transported to the cell surface. These results suggest that one function of the Golgi matrix is to aid efficient retention or sequestration of p24 cargo receptors and other membrane proteins in the Golgi apparatus.     

Membrane targeting of p115 phosphorylation mutants and their effects on Golgi integrity and secretory traffic

The cytosolic phosphoprotein p115 is required for ER to Golgi traffic and for Golgi reassembly after mitosis. In cells, p115 is localized to ER exit sites, ER-Golgi Intermediate Compartment (ERGIC) and the Golgi, and cycles between these compartments. P115 is phosphorylated on serine 942, and this modification appears to control p115 association with membranes. P115 is likely to function by reversibly interacting with effector proteins, and in the Golgi, two proteins, GM130 and giantin, have been shown to bind p115. The GM130-p115 and the giantin-p115 interactions are enhanced by p115 phosphorylation. Phosphorylation appears to be essential for p115 function, since substitutions of serine 942 abolish p115 ability to sustain cisternal reformation in an in vitro assay reconstituting Golgi reassembly after mitosis. Here, we explored how phosphorylation of p115 affects its intracellular targeting to distinct cellular compartments, and its function in secretory traffic. We generated phosphorylation mutants of p115 and tested their ability to target to ER exit sites, ERGIC and the Golgi. In addition, we explored whether expression of the mutants causes disruption of Golgi structure and perturbs ER-Golgi traffic of a VSV-G cargo protein.