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Originally published In Press as doi:10.1074/jbc.M412553200 on November 30, 2004

J. Biol. Chem., Vol. 280, Issue 6, 4442-4450, February 11, 2005
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Cell-free Transport from the trans-Golgi Network to Late Endosome Requires Factors Involved in Formation and Consumption of Clathrin-coated Vesicles*

Mohamed E. Abazeed, Jennifer M. Blanchette, and Robert S. Fuller{ddagger}

From the Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109

Received for publication, November 5, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transport between the trans-Golgi network (TGN) and late endosome represents a conserved, clathrin-dependent sorting event that separates lysosomal from secretory cargo molecules and is also required for localization of integral membrane proteins to the TGN. Previously, we reported a cell-free reaction that reconstitutes transport from the yeast TGN to the late endosome/prevacuolar compartment (PVC) and requires the PVC t-SNARE Pep12p. Here, we report that factors required both for formation of clathrin-coated vesicles at the TGN (the Chc1p clathrin heavy chain and the Vps1p dynamin homolog) and for vesicle fusion at the PVC (the Vps21p rab protein and Vps45p SM (Sec1/Munc18) protein) are required for cell-free transport. The marker for TGN-PVC transport, Kex2p, is initially present in a clathrin-containing membrane compartment that is competent for delivery of Kex2p to the PVC. A Kex2p chimera containing the cytosolic tail (C-tail) of the vacuolar protein sorting receptor, Vps10p, is also efficiently transported to the PVC. Antibodies against the Kex2p and Vps10p C-tails selectively block transport of Kex2p and the Kex2-Vps10p chimera. The requirements for factors involved in vesicle formation and fusion, the identification of the donor compartment as a clathrin-containing membrane, and the need for accessibility of C-tail sequences argue that the TGN-PVC transport reaction involves selective incorporation of TGN cargo molecules into clathrin-coated vesicle intermediates. Further biochemical dissection of this reaction should help elucidate the molecular requirements and hierarchy of events in TGN-to-PVC sorting and transport.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The trans-Golgi network (TGN)1 functions as a central processing compartment that modifies and sorts newly synthesized proteins destined for the plasma membrane, the endosomes, and the lysosome/vacuole (1). A study of the transport events between the TGN and these various compartments is fundamental to understanding molecular sorting in the distal arm of the biosynthetic pathway. TGN-to-late endosome transport serves at least two known trafficking functions in eukaryotes from yeast to humans. First, vesicle-mediated transport between the TGN and the late endosome is an important intermediate step in lysosomal/vacuolar biogenesis, facilitating transport of hydrolytic enzymes destined for the lysosome (2). Second, TGN-to-late endosome transport functions in the localization of TGN integral membrane proteins; a function critical in maintaining the processing and sorting activities of the TGN (3).

Similar to the pathway of lysosomal biogenesis in mammalian cells, inactive precursors of vacuolar enzymes in yeast are recognized in the TGN by the Vps10p transmembrane receptor, which delivers the cargo enzymes to the prevacuolar compartment (PVC) by a clathrin-dependent vesicle intermediate (4). After reaching the PVC, the hydrolytic enzymes are delivered to the vacuole where they are activated, and the receptors are recycled back to the TGN for another round of transport.

TGN-to-PVC transport represents the forward step in a highly regulated bi-directional, vesicle-mediated exchange between these two compartments. TGN integral membrane proteins, such as the proprotein processing enzymes Kex2p and Ste13p, cargo-sorting receptor Vps10p, and components of the vesicle fusion machinery, require continual cycling between the TGN and the late endosome for proper function and stability (5–8). Interruption in this cycling mechanism causes the mislocalization of TGN resident proteins, with consequent loss of processing and sorting activities in the TGN.

In yeast, fusion of TGN-derived transport vesicles with the PVC requires Pep12p, a PVC-localized t-SNARE (9–11). Previously, we characterized a cell-free reaction in which membranes containing the proprotein processing protease, Kex2p, fuse with membranes containing a PVC-localized Pep12p chimera (12) (Fig. 1A). This reaction exhibited characteristics of SNARE-dependent membrane fusion, including dependence on Pep12p. Here, we offer a detailed biochemical analysis of this TGN-to-PVC transport reaction to identify the precise mechanism by which Kex2p and Pep12p compartments undergo fusion in cell extracts. We find that molecules required for both formation of clathrin-coated vesicles at the TGN, the Chc1p clathrin heavy chain and the Vps1p dynamin homolog, and vesicle fusion at the PVC, the Vps21p rab protein, and Vps45 SM (Sec1/Munc18) protein, are required for cell-free transport. These results offer strong evidence supporting the hypothesis that fusion between Kex2p- and Pep12p-containing membranes in this system represents authentic TGN-to-PVC transport. Beyond this validation phase, we show that Kex2p molecules competent for delivery to the PVC reside initially in a clathrin-containing compartment. In addition, we show that a Kex2p chimera containing the cytosolic tail (C-tail) of the vacuolar protein sorting receptor, Vps10p, is also efficiently transported to the PVC, allowing us to now study the direct transport of two TGN transmembrane proteins that exhibit different trafficking itineraries (13). Finally, we show antibodies against the C-tails of Kex2p and Vps10p selectively block transport of Kex2p and the Kex2-Vps10p chimera, consistent with the interpretation that these TGN proteins must be incorporated into a coated vesicle in order to reach the PVC. Taken together, these results argue that delivery of TGN proteins to the PVC in this system is a cargo-selective process that proceeds through a clathrin-coated vesicular intermediate.



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  		FIG. 1.
Experimental design of cell-free TGN-to-PVC vesicle-mediated transport assay and cytosol dependence. A, schematic depiction of Kex2p transport from TGN to PSHA-containing PVC. The structure of the Pep12-Ste13{Delta}TMD{alpha}(3xHA) chimera protein (PSHA) (12) is depicted as well. In PSHA, the full-length Pep12p sequence is fused to the luminal portion of Ste13{Delta}TMD (Ste13p lacking cytosolic region and transmembrane domain), which in turn has a Kex2p cleavage sequence (taken from pro-{alpha}-factor) followed by a triple HA epitope tag fused at the C terminus. B, cytosol dependence. Isolated HSP Kex2p and PSHA membranes were combined in reactions containing indicated amounts of cytosol and incubated at 30 °C for 60 min. MSS membrane control (left-most bar) was carried out with equivalent amounts of membranes based on Kex2 and Ste13 enzymatic activity. 9.2% of DPAP processed was adjusted to represent 100% of transport.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Plasmids—Yeast strains and plasmids used in this study are listed in Table I; their construction is described below. JBY209 and BLY9 were as described (14). BLY26 was generated by transforming JBY209 with plasmid pRS314vps45-37ts expressing TRP1 and subsequently deleting the chromosomal VPS45 gene by the PCR method using the URA3 gene from pUG72 as a marker. PCR primers were designed with homology to 45 nucleotides upstream and down-stream of the VPS45 structural gene. vps45{Delta} was confirmed by colony PCR. The vps1-100 temperature-sensitive allele was introduced by transplacement (15). JBY209 was transformed with plasmid pCAV40 linearized with EcoRI and Ura+ colonies selected. MAY1 was a Ura– loop-out (selected on 5-fluoroorotic acid plates) that exhibited temperature-sensitive growth. The chc1-521ts allele was introduced, similarly, by transforming JBY209 with YIpchc521-{Delta}Cla linearized with XbaI, selecting Ura+ colonies. MAY2 was a Ura– loop-out (selected on 5-fluoro-orotic acid plates) that exhibited temperature-sensitive growth. pRS314vps45-37ts was constructed by inserting a ApaI/SacI fragment containing vps45-37ts allele into vector pRS314. A gene encoding the Kex2-Vps10p chimera was constructed by replacing codons 698 and 699 in KEX2 and codons 1416 and 1417 in VPS10 with an NdeI site (CATATG) and replacing sequences encoding the Kex2p C-tail with the NdeI-BstYI fragment encoding the Vps10p C-tail. The predicted amino acid sequence across chimeric junction, Met-Phe-His-Met-Gly-Ile, was confirmed by DNA sequencing. A plasmid encoding a glutathione S-transferase (GST)-Vps21p fusion, pYEX4T1-VPS21, was isolated from an ordered array (16). Plasmid pSN218 encoding HA epitope-tagged Kex2p was a gift of S. Nothwehr (University of Missouri, Columbia, MO).


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  		TABLE I
Strains and plasmids in this study

 
Antibodies and Reagents—Affinity-purified anti-Kex2p antiserum was as described (17). Anti-HA monoclonal antibody 12CA5 was from Roche Applied Science (Indianapolis, IN). Antisera against Chc1p and Clc1p were gifts from S. Lemmon (Case Western Reserve University) and R. Schekman (University of California, Berkeley). Anti-Vps10p C-tail monoclonal antibody was from Molecular Probes (Eugene, OR). Glutathione-Sepharose 4B and protein A-Sepharose beads were from Amersham Biosciences (Piscataway, NJ). Bovine {alpha}-thrombin was from Hematologic Technologies, Inc. (Essex Junction, VT). Restriction and DNA modification enzymes were from New England Biolabs (Beverly, MA). Other chemicals and reagents were from Sigma-Aldrich.

Membrane and Cytosol Preparations—Medium-speed supernatant (MSS) membranes, high speed pellet (HSP) membranes, and high speed supernatant (HSS) cytosol were prepared from semi-intact yeast cells as described (14, 18).

Cell-free Transport—Cell-free transport reactions containing MSS membranes were performed essentially as described (12). Permeabilized cells were prepared from JBY209 containing pCWKX10 or pPEP12STE13{Delta}TMD{alpha}3xHA (PSHA). Frozen spheroplasts (200 µl) were thawed (25 °C, 2 min) and centrifuged (5 min, 14,000 rpm), and MSS fractions were collected. MSS isolated under these conditions has been previously shown to contain cytosol and late Golgi and endosomal membranes but not ER or early Golgi vesicles (14). For cell-free fusion, 10 µl of MSS membranes from both the Kex2p- and PSHA-expressing strains were added to 10 µl of 3x reaction mix (0.2 M sorbitol, 10 mM Hepes, pH 7, 75 mM KOAc, 4 mM MgOAc, 0.25 mM EGTA, 140 mM phosphocreatine, and 0.375 mg ml–1 creatine kinase) on ice. Reactions were started by shifting to 30 °C and unless indicated otherwise were incubated for 20 min. 10 µl were then removed and added to tubes containing either immunoprecipitation mix (1% Triton X-100, 1 mM EDTA, 20 µl of pansorbin, 1 µl of 12CA5 monoclonal anti-HA, and 1 µl of rabbit anti-mouse IgG) or mock IP mix (1% Triton X-100, 1 mM EDTA, 20 µl of pansorbin, and 2 µl of water). Immunoprecipitates were incubated at room temperature with gentle agitation for 30 min. Pansorbin was pelleted, and 30 µl of each supernatant fraction were assayed for DPAP activity. The fraction of PSHA processed was calculated as the ratio of DPAP activity in the supernatant fraction (i.e. the activity that was immunodepletion-resistant) to the DPAP activity in the mock immunoprecipitation reaction (i.e. the total). Typically, incubation of PSHA and Kex2p MSS resulted in 10–12% of DPAP remaining in the supernatant after immunoprecipitation. Incubation of PSHA MSS with kex2{Delta} MSS resulted in 1–3% of DPAP remaining in the supernatant after immunoprecipitation. Tests for temperature-sensitive protein requirements were conducted by preincubating membranes at the permissive (25 °C) or non-permissive (35 °C) temperatures in the absence of reaction mix. After preincubation, reaction mix (0.2 M sorbitol, 10 mM HEPES pH 7, 75 mM KOAc, 4 mM MgOAc, 0.25 mM EGTA, 140 mM phosphocreatine, 0.375 mg/ml creatine kinase) was added on ice, and reactions were initiated by shifting to 25 °C and incubated for 20 min prior to processing as described (14). Data points represent mean values of duplicates; all reactions were performed at least twice with comparable results. Error bars represent the S.D. of the average of at least two reactions.

Preparation of Soluble Vps21p—A GST-Vps21p fusion protein, with a free C terminus to permit in vivo geranyl-geranylation, was expressed in yeast strain JBY209 from plasmid pYEX4T1-VPS21 and the protein was purified. Two hours prior to harvest, GST-Vps21p expression was induced by adding 100 µM CuSO4 to activate the CUP1 promoter on pYEX4T1. A control culture did not receive copper. Extract prepared from induced and uninduced cells by spheroplast lysis was incubated with glutathione-Sepharose 4B beads for 30 min at room temperature. Beads were washed 3x (10 ml) with phosphate-buffered saline buffer and GST fusion protein bound to beads was cleaved by bovine {alpha}-thrombin to release Vps21p. Thrombin was inactivated with 1 mM phenylmethylsulfonyl fluoride, and the soluble fraction and three 1-ml washes of thrombin-treated beads were pooled, concentrated, and dialyzed against PBS buffer. A Coomassie-stained gel of the purified fraction from induced cells showed substantial enrichment of the Vps21p to about 50% purity. A Coomassie-stained gel of the mock-purified fraction from uninduced cells showed no Vps21p band.

Subcellular Fractionation—JBY209 cells were grown at 30 °C to an OD600 of 1.0 in YPD and collected by centrifugation at 5000 x g for 10 min. Spheroplasts, generated by lyticase treatment, were frozen over liquid N2. Frozen spheroplasts (200-µl samples equivalent to 40 ml of cells at OD600 = 1.0) were thawed (25 °C, 2 min) and centrifuged (5 min, 14,000 x g), and the MSS fraction containing microsomes and cytosol was collected. To separate membranes from cytosol, MSS membranes were centrifuged at 200,000 x g in a TLS55 rotor (Beckman Coulter) for 1 h at 4 °C to generate HSP membranes and HSS cytosol. Samples equivalent to 2 ml of cells (OD600 = 1.0) of the medium speed pellet (MSP), MSS, HSP, and HSS were fractionated by SDS-PAGE, and the presence analyzed by immunoblotting.

Immunoisolation of Clathrin-containing Membranes—Immunoisolation of clathrin-containing membranes proceeded as described (19) with the following exceptions. Cells were converted to spheroplasts by lyticase treatment and lysis was induced by a slow freeze over liquid N2 vapors followed by a rapid thaw at 25 °C for 2 min (18). MSS generated by centrifugation for 5 min at 14,000 x g was gently layered on a 200-µl cushion of 80% Percoll and centrifuged at 200,000 x g in a TLS55 rotor (Beckman Coulter) for 1 h at 4 °C. The supernatant was discarded, and the pellet fraction was resuspended in 500 µl of MES buffer (100 mM MES, pH 6.5, 0.2 M sorbitol, 0.5 mM MgCl2, 1 mM EGTA, 0.2 mM dithiothreitol). 20 µl of antiserum against Clc1p were added to resuspended membrane preparations diluted in 1 ml of MES buffer containing 1% bovine serum albumin. These mixtures were incubated for 2 h at 4 °C with constant rotation. Immunocomplexes were isolated by addition of 30 µl of protein A-Sepharose (20% in MES buffer) and by incubation for an additional hour at 4 °C. Protein A-Sepharose pellets were subsequently washed and eluted in SDS-PAGE sample buffer. Eluted samples were fractionated by SDS-PAGE and analyzed by immunoblotting.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
TGN-to-PVC Membrane Fusion Is Cytosol-dependent— Elsewhere, we have reported a cell-free fusion reaction between Kex2p- and Pep12p-containing MSS membranes released from semi-intact yeast cells that exhibited specific biochemical and molecular requirements (12). In this reaction, fusion of MSS membranes containing Kex2p with MSS membranes containing a Pep12p fusion protein, PSHA (see Fig. 1A for description), results in cleavage of PSHA by Kex2p, monitored by enzymatic activity of Ste13 dipeptidyl aminopeptidase released in the soluble phase of an immunoprecipitation (14). To determine whether this reaction exhibited characteristics expected of TGN-to-PVC transport in vivo, we examined specific requirements for proteins involved in clathrin-coated vesicle formation at the TGN and vesicle fusion at the PVC. Vesicle formation, targeting and fusion depend on recruitment of cytosolic components to membranes. To assess the cytosol dependence of the reaction, Kex2p and Pep12p membranes were pelleted at 200,000 x g (P200) through a 12.5% Ficoll cushion and resuspended in membrane extraction buffer. HSP incubated under standard reaction conditions exhibited poor processing of the PSHA substrate (Fig. 1B). The addition of cytosol (HSS) to the membrane fractions restored fusion in a concentration-dependent fashion. The reconstituted reaction reached completion in 60 min (data not shown) and thus was slower than the reaction using unfractionated membranes, which reaches maximal level of PSHA processing after 20 min of incubation (12). However, the extent of the reconstituted reaction (80% of the control) was similar to the reaction using unfractionated membranes.

Requirement for Rab Vps21p and SM Protein Vps45p—In vivo studies indicate that the Rab GTPase protein Vps21p is involved in fusion of Golgi-derived transport vesicles with the PVC (20, 21). Membranes from vps21 mutant cells should therefore be defective for cell-free TGN-PVC transport. Membranes from a vps21 mutant strain expressing either Kex2p or PSHA were prepared and tested for fusion. Although there was a modest reduction in the level of PSHA cleavage when membranes from the Kex2p vps21 strain were used, a severe defect was observed when the PSHA-containing membranes were prepared from a vps21 mutant strain (Fig. 2A). Absence of Vps21p function from both membrane preparations resulted in a further loss of fusion competence (Fig. 2A).



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  		FIG. 2.
Vps21p is required for TGN-to-PVC transport. A, Kex2p and PSHA MSS membranes from VPS21 strain JBY209 and vps21 mutant strain BLY9 were combined in transport assays as indicated. Reactions were incubated at 30 °C for 20 min. Data points were calculated as a percentage of PSHA cleavage in the reaction in which both the Kex2p and PSHA membranes were from JBY209. 10.3% of DPAP processed was adjusted to represent 100% of transport. B, purified, wild-type Vps21p from cells induced with 100 µM CuSO4 was added to MSS from the vps21 mutant strain as indicated. Right hand bar, control reaction with mock-purified sample from uninduced cells (no CuSO4). Reactions were incubated at 30 °C for 60 min. 12.4% of DPAP processed was adjusted to represent 100% of transport. C, MSS membranes were incubated with 0.5 mM Latrunculin B in methanol or an equivalent volume of methanol on ice for 60 min and then tested for the recovery of transport by adding purified Vps21p. Reactions were incubated at 30 °C for 60 min. 11.5% of DPAP processed was adjusted to represent 100% of transport.

 
Although it was evident that the lack of Vps21p function on PSHA-containing membranes led to the majority of loss of fusion activity, it was not clear whether this loss was due to missorting of the PSHA substrate in vivo or whether the defect was due directly to the inability of Golgi-derived vesicles to fuse with the Pep12p compartment. To distinguish between these possibilities, wild-type Vps21p expressed in yeast as a GST fusion, purified from yeast extracts and cleaved from the GST tag from a Cu2+-inducible GST-VPS21 vector was purified from yeast as described under "Experimental Procedures" (16). Addition of purified Vps21p to MSS membranes from the vps21 mutant strain led to a recovery of membrane fusion that was concentration-dependent (Fig. 2B). The required time of incubation was similar to that needed for reconstitution of fusion using P200 membranes incubated with cytosol, suggesting that the delay in that experiment represents reassociation of protein factors with membranes. This experiment suggested that Vps21p was required for fusion of Kex2p and Pep12p membranes, but it was important to determine whether or not the vps21 mutation altered the localization of the PSHA substrate. Loss of Vps21p function in vivo might result in accumulation of PSHA in TGN membranes. To rule out the possibility that addition of purified Vps21p promoted Kex2p-dependent cleavage of PSHA through TGN homotypic fusion, which also requires Vps21p (14), we examined inhibition by the actin depolymerizing compound Latrunculin B, which blocks TGN homotypic fusion but not TGN-PVC transport (12). As shown in Fig. 2C, Latrunculin B did not inhibit PSHA cleavage in vps21 membranes supplemented with purified Vps21p (Fig. 2C). These observations indicate that cell-free fusion of Kex2p-containing membranes and PVC-localized PSHA is Vps21p-dependent.

Mutations in the class D VPS genes PEP12, VPS21, or VPS45 result in similar phenotypes; moreover, all three gene products are thought to be part of a multiprotein complex that mediates tethering and fusion of TGN transport vesicles with the PVC (22). VPS45 encodes a Sec1/Munc18 (SM) protein that binds directly to and regulates the syntaxin Pep12p (23). Moreover, Vps45p is selectively involved in mediating entry of TGN-derived cargo proteins into the PVC and is not required for entry of endocytic cargo (24). Involvement of Vps45p in cell-free fusion of Kex2p and Pep12p membranes would thus provide additional evidence that delivery of Kex2p to the Pep12p membranes represents authentic TGN-PVC transport. This is important because although Kex2p is mainly localized at the TGN at steady state (17), Kex2p cycles through both the early endosome (EE), and PVC (13). Therefore, fusion between Kex2p and Pep12p membranes in the cell-free assay could represent TGN-to-PVC transport, EE-to-PVC transport, or a combination of these events. To distinguish these possibilities, we prepared membrane extracts from a vps45ts strain expressing either Kex2p or the PSHA substrate. The use of the conditional vps45 allele eliminated possible complications of in vivo missorting of Kex2p or Pep12p.

MSS membranes were prepared from the vps45ts strains grown at the permissive temperature (25 °C). Prior to the reaction, membranes were preincubated separately at 25 or 35 °C (non-permissive temperature) for 10 min. Preincubation at 35 °C had only a slight effect on the VPS45 wild-type membranes (Fig. 3A). When only the Kex2p-containing or PSHA-containing membranes were from the vps45ts background, preincubation at 35 °C resulted in a partial reduction (50–60%) in PSHA cleavage relative to that observed with membranes preincubated at 25 °C (Fig. 3A). PSHA cleavage was reduced to background levels (i.e. observed with kex2{Delta} membranes) when both Kex2p and PSHA membranes were from vps45ts strains and were preincubated at 35 °C (Fig. 3A). These results indicate that inactivation of the temperature-sensitive form of Vps45p blocks cell-free fusion of Kex2p and Pep12p membranes. Recently it has been demonstrated that Vps45p associates and disassociates from membranes at different stages of SNARE complex assembly (25), suggesting that vesicle docking regulates Vps45 recruitment to membranes. To determine whether Vps45p was recruited to membranes from a soluble pool in the cell-free reaction, vps45ts membranes inactivated by preincubation at 35 °C were pelleted at 200,000 x g. These membranes were then used in reconstituted transport reactions with VPS45 or vps45ts cytosol that had also been incubated for 10 min at 35 °C (Fig. 3B). Selective restoration of fusion was only observed with cytosol prepared from wild-type cells, suggesting that functional Vps45p from this fraction reassociates with membranes to regulate fusion. The kinetics of the reaction (completion at 60 min, data not shown) were similar to those observed in reconstitution of fusion using cytosol and purified Vps21p.



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  		FIG. 3.
Vps45p is required for TGN-to-PVC transport. A, Kex2p and PSHA MSS membranes from VPS45 strain JBY209 and vps45-37ts strain BLY26 were preincubated at 25 or 35 °C as described under "Experimental Procedures" and combined for transport reactions as indicated. Data points were calculated as a percentage of wild-type substrate processing for each preincubation temperature. 12.6% (25 °C) and 11.9% (35 °C) of DPAP processed were adjusted to represent 100% of transport for the corresponding preincubation temperature. B, after MSS membranes from BLY26 were preincubated at 35 °C for 10 min, HSP membranes were prepared. HSS cytosol fractions prepared from JBY209 and BLY26 were incubated at 35 °C for 10 min and added to the HSP membranes to measure recovery of fusion activity. Reactions were incubated at 25 °C for 60 min. Data points were calculated as a percentage of wild-type substrate processing. 11.4% of DPAP processed was adjusted to represent 100% of transport.

 
Vps1p, Chc1p, and Vesicle Formation at the TGN—The experiments above establish that Vps21p and Vps45p are recruited from the cytosol to regulate fusion of Kex2p and Pep12p containing membranes. To demonstrate that this reaction is a reconstitution of vesicle transport from the TGN-to-PVC, we examined the involvement of molecules thought to be required for formation of TGN-PVC transport vesicles at the TGN, focusing on the dynamin homolog Vps1p and on clathrin heavy chain, Chc1p.

Mutations in CHC1 result in defects in pro-{alpha}-factor processing and mislocalize to the plasma membrane proteins such as Kex2p, Ste13p, and Vps10p that normally cycle between the TGN and PVC (26, 27). These findings argue that, as in mammalian cells (28), clathrin-coated vesicles are involved in TGN-late endosome transport. Mutations in VPS1 result in defects both in sorting of the carboxypeptidase Y (CPY) precursor to the vacuole and in localization of TGN membrane proteins Kex2p and Ste13p (29). Like chc1 mutants, vps1 mutants also mislocalize Kex2p and Ste13p to the plasma membrane (15). The phenotypic similarity of chc1 and vps1 mutations, along with synthetic interactions between chc1 and vps1 alleles (30) suggest that Vps1p and Chc1p function together in formation of TGN-PVC transport vesicles at the TGN.

To determine whether Vps1p is required for cell-free TGN-PVC transport, we prepared MSS membranes from a strain carrying a temperature-sensitive VPS1 allele, vps1-100ts. As in the case of the vps45ts strain, membranes were prepared from cells grown at 25 °C with all subsequent steps at or below that temperature to avoid effects on Kex2p and Pep12p sorting in vivo. Prior to initiating reactions, wild-type and vps1-100ts membranes were separately preincubated at 25 or 35 °C for 10 min. Preincubation of the vps1-100ts membranes at 35 °C resulted in a substantial reduction in PSHA cleavage (Fig. 4A), suggesting the involvement of Vps1p in the cell-free fusion of Kex2p and Pep12p membranes.



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  		FIG. 4.
Vps1p and Chc1p are required for TGN-to-PVC transport. A and B, Kex2p and PSHA MSS membranes were prepared from vps1-100ts strain MAY1 and chc1-521ts strain MAY2. MSS membranes were preincubated at 25 °C or 35 °C as described under "Experimental Procedures" and combined for transport reactions as indicated. Data points were calculated as percent of wild-type control (VPS1 or CHC1) for each preincubation temperature. 10.4% (25 °C), 9.6% (35 °C) VPS1, and 10.1% (25 °C), 9.7% (35 °C) CHC1 of DPAP processed were adjusted to represent 100% of transport. C, monoclonal anti-Chc1p antibodies ({alpha}-Chc1p, 2.5 µg) were incubated with Kex2p and PSHA MSS membranes for 1 h at 4 °C. Reactions were then incubated at 30 °C for 20 min. 10.7% of DPAP processed were adjusted to represent 100% of transport.

 
To test for clathrin involvement in this reaction, we prepared MSS membranes from a chc1-521ts strain and tested them for fusion. Preincubation of the chc1-521ts membranes at 35 °C resulted in a ~3-fold reduction in PSHA cleavage (Fig. 4B), implying a role for clathrin in the cell-free fusion of Kex2p and Pep12p membranes. Furthermore, addition of a mixture of monoclonal anti-clathrin heavy chain antibodies (31) to a reaction containing WT Chc1p resulted in 60% inhibition of the reaction (Fig. 4C). Requirements for both Vps1p and Chc1p argue strongly that the fusion of Kex2p- and PSHA-containing membranes involves formation of clathrin-coated vesicles at the TGN.

Immunoisolation of Kex2p-containing Clathrin-associated Membranes—Identification of clathrin as the putative coat protein in cell-free TGN-PVC fusion and the observed effects of clathrin mutations on Kex2p localization (32) led us to examine whether clathrin is bound to the cytosolic surface of Kex2p-containing MSS membranes. Clathrin is composed of three heavy chains (Chc) and three light chains (Clc) that are arranged in trimeric complex (triskelion) (33). Initially, we examined the degree of association of clathrin with MSS membranes by subcellular fractionation experiments. Lysates of freeze-thaw lysed spheroplasts of the Kex2p-expressing strain were first fractionated by differential centrifugation. The lysate was centrifuged at 14,000 x g, generating the standard MSS used in the assay and MSP. MSS isolated under these conditions has been previously shown to contain cytosol and late Golgi and endosomal membranes but not ER or early Golgi vesicles (14). MSS was then centrifuged at 200,000 x g to generate HSS and pellet (HSP) fractions, separating microsomes in the HSP from soluble cytosolic factors in the HSS. Immunoblot analysis of the fractions using anti-Chc1p antibody demonstrated that the majority of Chc1p in the MSS was in the cytosolic fraction (HSS), with only a small amount (~5%) remaining in the microsomal fraction (Fig. 5A). This suggested that at the initial stages of the reaction, only a small fraction of total clathrin was bound to membranes.



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  		FIG. 5.
Isolated clathrin-containing membranes act as Kex2p donor compartment. A, subcellular fractionation was performed as described under "Experimental Procedures" to determine Chc1p localization. B, MSS membranes from strain JBY209 transformed with plasmid pSN218 encoding HA-tagged Kex2p (15) were tested for competence in substituting for wild-type Kex2p in the standard cell-free TGN-PVC assay. C, HSP membranes as prepared in A were subjected to immunopurification by addition of anti-Clc1p as described under "Experimental Procedures." Lane 1 represents 10% of the amount of HSP subjected to isolation. Lane 2, immunoisolate from CHC1 strain JBY209 transformed with pSN218. Lane 3, immunoisolate from chc1-521ts strain MAY2 transformed with pSN218 and shifted to 37 °C 30 min prior to harvest. D, immunoisolate shown in C was tested for competence in transport using MSS membranes prepared from JBY209 expressing PSHA. Mock indicates reaction performed with a sample prepared by mock-isolation using protein A-Sepharose incubated with HSP membranes without the addition of anti-Clc1p. Reactions were incubated at 30 °C for 60 min. Results are representative (n = 3).

 
To determine whether the Chc1p membranes in the HSP fraction also contained Kex2p, we isolated the clathrin-associated membranes in the HSP fraction using antibodies against clathrin-light chain, Clc1p, (19, 34) and probed for the presence of Kex2p. An epitope-tagged form of Kex2p, Kex2-HAp, shown previously to fully complement kex2{Delta} mutations for processing pro-{alpha}-factor (15), was used in these experiments. Kex2-HAp, introduced into the standard fusion strain, JBY209, was fully functional for PSHA cleavage in the cell-free reaction (Fig. 5B). MSS prepared from this strain was centrifuged at 200,000 x g, and sedimented membranes were collected onto a 200-µl cushion of 80% Percoll. The HSS fraction was discarded and the HSP microsomes were collected and immunoprecipitated with rabbit anti-Clc1p antiserum. Precipitates were analyzed by SDS-PAGE and immunoblotted using anti-Chc1p and anti-HA antibodies (Fig. 5C). Consistent with previous studies (19), Chc1p was found to co-precipitate with anti-Clc1p antibodies (Fig. 5C, lane 3). The isolation of clathrin heavy chain from a high speed microsomal fraction suggested that the clathrin was bound to the membrane surface. Kex2-HAp was also co-immunoisolated (Fig. 5C, lane 2), demonstrating that at the onset of the reaction, the majority of Kex2p was present in a clathrin-containing membrane compartment. To test the specificity of the anti-Clc1p immunoisolation procedure, we examined the effects of the chc1-521ts mutation on recovery of both Chc1p and Kex2p. For this purpose, chc1-521ts cells were grown at 24 °C, converted to spheroplasts, and shifted to 37 °C 30 min prior to freeze-thaw lysis. Lane 3 in Fig. 5C demonstrates that the Clc1p antibody failed to co-immunoprecipitate either Chc1p or Kex2-HAp, implying that the association of clathrin light and heavy chains is required for the immunoisolation of Kex2p-containing membranes by anti-Clc1p antibodies.

The localization of Kex2p within clathrin-associated membranes at the beginning of the reaction and the dependence of the reaction on clathrin suggested that the clathrin-containing membranes serve as the Kex2p "donor" compartment in the reaction. To demonstrate this directly, clathrin-coated membranes immunoisolated using anti-Clc1p antibodies were incubated with MSS membranes containing the PSHA substrate (Fig. 5D). Because immunoprecipitates were found to contain ~40% of the amount of Kex2p used in a standard transport reaction (based on enzymatic activity of Kex2p, data not shown), two anti-Clc1p immunoprecipitates were combined and subsequently incubated with PSHA MSS membranes. PSHA cleavage was observed with immunoisolated membranes as donor but not with the mock-immunoprecipitation control, confirming that clathrin-associated membranes can serve as Kex2p donor membranes in the reaction.

Cytosolic Tail Dependence and Vps10p Transport—Vps10p and Kex2p cycle between the TGN and the PVC and both proteins contain signals in their cytosolic tails that mediate their selective transport between these compartments (35–38). Unlike Vps10p, Kex2p cycles through an early endosomal (EE) compartment as well as through the PVC (13). To determine whether the TGN rather than the EE represents the Kex2p donor compartment in the cell-free assay, we sought to establish an assay that measures Vps10p transport from the TGN to the PVC. For this purpose, a Kex2-Vps10p chimera was constructed that contained the N-terminal luminal and transmembrane domains of Kex2p fused to the C-tail domain of Vps10p (Fig. 6A). PSHA substrate cleavage observed with Kex2-Vps10p as the sole source of Kex2p activity in the donor compartment was equivalent to that seen with wild-type Kex2p (Fig. 6B). Because Vps10p is delivered directly from the TGN to the PVC, the behavior of the Kex2-Vps10p chimera suggests that the TGN rather than the EE is the donor compartment in this reaction.



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  		FIG. 6.
Kex2p and Vps10p C-tails are required for TGN-PVC transport. A, domain composition of Kex2p, Vps10p, and chimeric Kex2p, Kex2-Vps10p. Each segment represents one domain, starting with the N-terminal luminal domain, followed by the transmembrane domain and the C-terminal cytoplasmic domain. The numbers above give the N- and C-terminal amino acids of each domain. For the chimeric receptor the amino acids representing the junction between domains are given (see "Experimental Procedures" for details on chimera construction). B, MSS membranes from JBY209 expressing Kex2p or the Kex2-Vps10p chimera (under "Experimental Procedures") were tested for cell-free TGN-PVC transport. Reactions were incubated at 30 °C for 20 min. C, affinity-purified anti-Kex2p C-tail antibodies ({alpha}-Kex2p Tail, 1.7 µg) and monoclonal anti-Vps10p C-tail antibodies ({alpha}-Vps10p Tail, 1.5 µg) were incubated with Kex2p and Kex2-Vps10p membranes for 1 h at 4°C. Competence for TGN-PVC transport was then tested using PSHA MSS membranes. Reactions were incubated at 30 °C for 20 min. 9.4% (Kex2p) and 12.7% (Kex2-Vps10p) of DPAP processed were adjusted to represent 100% of transport. D, this schematic summarizes the positional and functional roles of Pep12, Vps21p, Vps45p, Vps1p, Chc1p, and C-tail domains in TGN-to-PVC vesicle transport.

 
It has been established that the C-tail domain of Vps10p, although required for retrieval from the PVC, is not required for recruitment into clathrin coated vesicles (19, 37). Moreover, the C-tail of Kex2p is not required for clathrin-dependent transport of Kex2p to the vacuole (39). Although these data suggest that the C-tails are not essential for the entry of cargo into vesicles, they do not preclude the existence of signals in the Kex2p and Vps10p C-tails that facilitate entry of the proteins into clathrin-coated vesicles. Indeed, Pep12p contains a signal that directs its GGA-dependent transport from the TGN to the PVC (40). Moreover sorting of the general amino acid permease, Gap1p, from the TGN to the PVC is regulated by ubiquitinylation of cytosolic sequences (41). We therefore asked whether antibodies against the Kex2p and Vps10p C-tails would interfere with cell-free TGN-PVC transport. An affinity-purified antibody against the C-tail of Kex2p specifically reduced the extent of PSHA cleavage when wild-type Kex2p was present in donor membranes but not when membranes containing the Kex2-Vps10p chimera were used (Fig. 6C). Conversely, monoclonal antibody against the Vps10p tail inhibited PSHA cleavage when the Kex2-Vps10p chimera was present in donor membranes but only had a slight effect when membranes containing wild-type Kex2p were used (Fig. 6C). These experiments do not distinguish whether the C-tail antibodies inhibit by a steric mechanism or by obscuring sorting information in the C-tails. However, in either case, the antibodies likely target the cargo recruitment step of vesicle formation, further supporting our model that Kex2p and PSHA membrane fusion in this system represents vesicle transport from the TGN to the PVC. All together, the inhibition of substrate processing by manipulations that interfere with factors (Vps1p, Chc1p, C-tails) that mediate vesicle formation provides strong evidence that active vesicle budding occurs during cell-free TGN-PVC transport.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Previous observations have led to a model in which steady-state localization and stability of Kex2p and other TGN transmembrane proteins is maintained by cycling between the TGN and the PVC. The necessity for such a complex mechanism of localization is evident from the dynamic nature of secretory organelles such as the TGN. The TGN itself can be thought of as the terminal phase in maturation of Golgi cisternae, breaking down into secretory carrier vesicles and vesicles that carry TGN components to the endosomal system (42). The delivery to and subsequent retrieval of TGN membrane proteins from the PVC/late endosome serve both to drive vacuolar/lysosomal biogenesis and to restore processing molecules to newly forming TGN.

In an effort to establish a model system for studying TGN-to-PVC vesicle traffic, we have reconstituted this transport event in a cell-free assay. The molecular characterization of this reaction establishes direct involvement of proteins that mediate membrane fusion at the PVC and those mediating vesicle formation at the TGN, arguing that fusion of Kex2p and Pep12p membranes occurs by vesicle mediated TGN-to-PVC transport (Fig. 6D). Moreover, the requirement for clathrin and the co-localization of clathrin and Kex2p in a membrane compartment that is competent for delivery of Kex2p to the PVC confirms that clathrin is the coat protein responsible for Kex2p transport and suggests the de novo emergence of a clathrin vesicle intermediate in the course of the reaction. The same enzymatic system mediates delivery of a chimeric Kex2, containing the cytosolic tail of Vps10p, from the TGN-to-PVC. The ability to directly measure the transport of two distinct cytosolic tail signals from the TGN into the PVC will enable us to identify differences in the mechanisms of molecular transport between Kex2p and Vps10p. Preliminary results suggest intriguing roles for the adaptors AP1 and GGA in mediating the trafficking of these signals.

Based on the molecular requirements we have demonstrated, this system appears to recapitulate all phases of TGN- to-PVC transport including cargo recruitment and budding of clathrin-coated vesicles, vesicle fission, uncoating of the transport vesicles, and targeting of the vesicles to and fusion with PVC membranes. Consequently, this system makes possible analysis of the biochemical mechanisms of each of these steps. The requirement for clathrin predicts that a clathrin-coated vesicle is an intermediate in the reaction. This can be tested by examining the evolution and consumption of such vesicles during the reaction (43). The involvement of clathrin raises the question of which adaptor proteins are required for clathrin recruitment and whether Arf1p GTPase is involved. The nature of interactions, if any, between signals in the Kex2p and Vps10p cytosolic tails with elements of the clathrin coat can be probed. A possible role for Vps1p, a member of the dynamin GTPase family, in vesicle fission can now be tested directly. The rab GTPase Vps21p may interact with multiple effectors in TGN-PVC transport. Identities and roles of these effectors should be accessible in this system. Although the role of the syntaxin Pep12p as a t-SNARE in TGN-PVC transport is well established, there has been no definitive characterization of the SNARE complex that forms during fusion of TGN-derived vesicles with PVC membranes. The nature of this complex should become directly accessible in the cell-free system; in particular, the recent suggestion that a Pep12p/Tlg1p/Vti1p t-SNARE pairs with Snc2p (44) can be tested directly. Finally, reconstitution of fusion with vps45 mutant membranes using wild-type but not vps45 mutant cytosol provides a system in which to probe the poorly understood mechanism of SM protein function in fusion.

Authentic reconstitution of the trafficking of integral membrane proteins that cycle between the TGN and PVC/late endosome is intrinsically difficult because such proteins are found in multiple compartments. In the absence of the experimental means to purify discrete organelles, the success of the cell-free approach hinges on devising an assay that isolates a specific transport event to be recreated in vitro. The selection of Kex2p as a donor membrane marker was made on the basis of its retention at the TGN and the EE, with transient cycling through the PVC. The use of the Kex2-Vps10p chimera is an experimental refinement that permits focus on biosynthetic pathway between the TGN and PVC. The choice of Pep12p as the marker for the acceptor compartment was driven by strong evidence for its functional role at the PVC and its concentration in this compartment. Application of similar criteria could lead to reconstitution of other key trafficking events that occur between the TGN and endosomal compartments.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants GM50915 and GM39697 (to R. S. F.), a University of Michigan Medical Scientist Training Program Grant GM0786 (to M. E. A.), the Genetics Training Program GM07544 (to M. E. A. and J. M. B.), a University of Michigan Rackham Graduate School predoctoral fellowship (to J. M. B.), and P30 CA46592 to the University of Michigan Comprehensive Cancer Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Biological Chemistry, 1301 E. Catherine Rd., University of Michigan, Ann Arbor, MI 48109-0606. Tel.: 734-936-9764; Fax: 734-763-7799; E-mail: bfuller{at}umich.edu.

1 The abbreviations used are: TGN, trans-Golgi network; C-tail, cytosolic tail; DPAP, dipeptidyl aminopeptidase; EE, early endosome; GST, glutathione S-transferase; HA, hemagglutinin; HSP, 200,000 x g pellet; HSS, 200,000 x g supernatant; MSS, medium speed supernatant; PSHA, Pep12Ste13{alpha}HA fusion substrate; PVC, prevacuolar compartment; SNARE, soluble NSF attachment protein receptor; TMD, transmembrane domain; t-SNARE, target membrane SNARE; v-SNARE, vesicle SNARE; MES, 4-morpholineethanesulfonic acid; ER, endoplasmic reticulum. Back


   ACKNOWLEDGMENTS
 
We thank S. Lemmon and R. Schekman for antibodies, S. Nothwehr, G. Payne, and S. Emr for strains and plasmids, and members of the Fuller laboratory for helpful comments on the manuscript.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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