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Originally published In Press as doi:10.1074/jbc.M406368200 on September 13, 2004

J. Biol. Chem., Vol. 279, Issue 47, 48767-48773, November 19, 2004
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Cell-free Reconstitution of Transport from the trans-Golgi Network to the Late Endosome/Prevacuolar Compartment*

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

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

Received for publication, June 8, 2004 , and in revised form, September 2, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Vesicle-mediated transport between the trans-Golgi network (TGN) and the late endosome/prevacuolar compartment (PVC) is an essential step in lysosomal/vacuolar biogenesis. In addition, localization of integral membrane proteins to the TGN requires continual cycles of vesicular transport between the TGN and endosomal compartments. Genetic and biochemical analyses in yeast have identified a variety of proteins required for TGN-to-PVC transport. However, the precise mechanisms of vesicle formation, transport, and fusion have not been fully elucidated. To study the steps of TGN-to-PVC transport in mechanistic detail, we have developed a cell-free assay to monitor delivery of the processing protease Kex2p from the TGN to PVC compartments containing a Kex2p substrate. Transport is time-, temperature-, and ATP-dependent and requires the t-SNARE Pep12p. Moreover, cell-free delivery of Kex2p to the PVC results in the co-integration of Kex2p into PVC membranes containing the Kex2p substrate as determined by co-immunoisolation of Kex2p and the substrate using antibody against the Kex2p cytosolic tail. This work represents the first cell-free reconstitution and biochemical analysis of the essential vacuolar/lysosomal sorting step TGN to late endosome transport.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Located at the intersection of the secretory, endocytic, and vacuolar/lysosomal protein-sorting pathways, the trans-Golgi network (TGN)1 presents an especially complex challenge for maintaining the specificity of protein trafficking. Anterograde protein cargo arriving at the TGN from earlier Golgi compartments and retrograde cargo proteins arriving from endocytic compartments must be sorted and packaged into distinct vesicles destined for one of at least four possible target compartments: the vacuole (yeast lysosome), the plasma membrane, the late endosome/prevacuolar compartment (PVC), or the early endosome. At the same time, the TGN must maintain the localization of resident transmembrane proteins such as Kex2p and Ste13p, proteases whose enzymatic activities define the TGN as a proprotein processing organelle (1, 2). Kex2p, Ste13p, and the procarboxypeptidase Y (CPY)-sorting receptor Vps10p all maintain steady-state TGN localization through cycles of transport between endocytic compartments and the TGN (3–5). Genetic and biochemical analyses have shown that clathrincoated vesicles mediate delivery from the TGN to the PVC (6–10). Retrieval of TGN transmembrane proteins from the PVC to the TGN, thought to be mediated by a distinct class of vesicles having a "retromer" coat, is signaled by aromatic residue-containing sequences in the cytosolic tails of the proteins (11). Additional proteins implicated in TGN-to-PVC transport include the dynamin GTPase homolog Vps1p (12), the adaptor proteins Gga1p and Gga2p (13–15), the class D VPS genes including the PVC-localized t-SNARE, Pep12p, the Sec1 homolog, Vps45p, and the rab GTPase Vps21p. In mammalian cells, the AP1 clathrin adaptor protein has also been implicated in this step (16). The precise mechanisms by which these proteins mediate vesicle formation at the TGN, transport, and fusion at the PVC remain elusive. In part, this is because of the difficulty of resolving rapid multistep processes by in vivo analysis.

Detailed biochemical analysis of interorganellar protein transfer has been facilitated by in vitro reconstitution of individual transport steps such as endoplasmic reticulum-to-early Golgi (17–20), intra-Golgi (21), late endosome-to-TGN (22, 23), and late endosome-to-vacuole transport (24). Here we report the development and biochemical analysis of a cell-free system for yeast TGN-to-PVC transport. The system involves combining membrane compartments harboring the TGN-localized Kex2 protease with membrane compartments containing an engineered PVC-localized Kex2p substrate in the presence of cytosol and an ATP-regenerating system. Delivery of Kex2p to the PVC is indicated by processing of the PVC-localized Kex2p substrate and has biochemical features consistent with authentic vesicular transport between the TGN and PVC including dependence on the PVC t-SNARE Pep12p.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and Reagents—Purified rabbit anti-Tlg2p IgG was as described (25). Monoclonal anti-Pep12p antibodies were from Molecular Probes, Inc. (Eugene, OR). Monoclonal anti-hemagglutinin (HA) antibody 12CA5 was from Roche, and rabbit anti-mouse IgG and affinity purified goat anti-rabbit IgG/Fc fragment-specific were from Jackson Laboratories (West Grove, PA). Other reagents were from Sigma unless indicated otherwise.

Strains and Plasmids—Strain JBY209 (MAT{alpha} ade2–1 can1–100 his3–11,15 leu2–3,112 trp1–1 ura3–1 kex2::hisG dap2::kanr pep4::HIS3 ste13::LEU2) and plasmids pCWKX10 and pSTE13{alpha}HA were as described (25). Strain BLY3 (JBY209 vps27–123ts) was generated by transforming JBY209 with Bcl1-digested, URA3-marked, vps27–123ts integrating plasmid, pRCP20 (26). To remove the wild-type copy of the VPS27 allele, URA3+ transformants were selected on 5-fluoroorotic acid (5-FOA), and a 5-FOAR isolate that demonstrated temperature-sensitive secretion of carboxypeptidase Y by colony immunoblotting was selected as BLY3. The diploid strain generated by crossing CBY9 (MAT{alpha} leu2–3,112 ura3–52 his3-{Delta}200 trp1-{Delta}901 lys2–801 suc2-{Delta}9 pep12–60tsf) (27) with JBY209a (JBY209 MATa) (25) was sporulated, and tetrads were scored to isolate BLY05 (JBY209 pep12–60tsf). Strain MAY21 (JBY209 pep12{Delta}::bleR) (pJB10) was generated by transforming JBY209 (pJB10) with a DNA fragment containing the phleomycin resistance gene from pUG66 (28) amplified using PCR primers having homology upstream and downstream of PEP12. Colony PCR and immunoblotting confirmed the deletion of PEP12.

To create the Kex2p substrate, PSHA, overlap extension PCR was used to fuse the entire PEP12 open reading frame to sequences encoding the Ste13{alpha}HA fusion protein (25) just beyond the Ste13p transmembrane domain (at nucleotide 448 of the STE13 sequence). The PSHA fusion gene was inserted into plasmid p416TEF (29) creating pJB10. Details of the construction are available on request. Plasmid pJB10-ts, encoding fusion protein PSHAtsf, was constructed in the same way as pJB10 except that the PEP12 portion was amplified from genomic DNA of yeast strain CBY9 (27).

Cell-free Transport—Medium speed supernatant containing TGN and endosomal membranes plus cytosol was prepared from semi-intact yeast cells as described (25, 30). Medium-speed supernatant membranes from Kex2p-expressing and either PSHA-, PSHAtsf-, or Ste13{alpha}HA-expressing strains were added (10 µl each) 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, 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 removed and added to tubes containing either immunoprecipitation (IP) mix (1% Triton X-100, 1 mM EDTA, 20 µl Pansorbin, 1 µl 12CA5 monoclonal anti-HA antibody, 1 µl rabbit anti-mouse IgG) or mock IP mix (1% Triton X-100, 1 mM EDTA, 20 µl Pansorbin, 2 µl water). IPs were incubated at room temperature with gentle agitation for 30 min. Pansorbin was pelleted, and 30 µl of each supernatant fraction were assayed for dipeptidyl aminopeptidase (DPAP) activity using substrate Ala-Pro-4-methyl-coumarin-7-amide as described (25). The fraction of PSHA processed was calculated as the ratio of DPAP activity in the supernatant fraction of the 12CA5-containing IP to that of the supernatant fraction of the mock IP. Error bars represent the standard deviation of the mean of at least two reactions.

Immunoisolation of Kex2p Membranes—Isolation of Kex2p-containing membranes using affinity-purified antibody to the cytosolic tail of Kex2p was carried out as described (31).

Preparation of Fab Fragments—Fab fragments of monoclonal anti-Pep12p IgG were prepared by digestion with papain as described (32). Papain digestion was confirmed by SDS-PAGE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of a Prevacuolar Compartment-localized Kex2p Substrate—Kex2p and Ste13p are both transmembrane-processing proteases localized at steady state to the yeast TGN where they mediate the proteolytic maturation of secretory proproteins such as pro-{alpha}-factor (33). Kex2p is a serine protease that specifically cleaves following paired basic amino acid motifs, whereas Step13p is a type IV dipeptidyl aminopeptidase with specificity for cleavage following X-Pro amino-terminal dipeptides. We described previously an assay for cell-free homotypic TGN fusion that measured the fusion of membranes containing Kex2p with membranes containing a Ste13p fusion protein, Ste13{alpha}HA, which functions as a reporter for Kex2-dependent cleavage (25). To develop an assay specific for TGN- to-PVC transport, a PVC-localized reporter (termed PSHA) was generated by fusing the luminal domain of Ste13{alpha}HA to the cytosolic and transmembrane domains of the PVC-localized t-SNARE, Pep12p (Fig. 1A) (13, 34). Pep12p is required at the PVC to mediate the fusion of incoming vesicular traffic; therefore, a loss of Pep12p function results in the accumulation of transport vesicles en route to the PVC (35, 36). Such a defect in delivery to the PVC causes the precursor of the vacuolar hydrolase carboxypeptidase Y to build up in the TGN and ultimately to be diverted into secretory vesicles (35). To determine whether PSHA was properly localized, the ability of PSHA expression to complement loss of PEP12 function was examined. Strains carrying the temperature-sensitive PEP12 allele, pep12tsf, and transformed with either a plasmid expressing PSHA or a vector control were assayed for CPY secretion at the nonpermissive temperature (Fig. 1B). The pep12–60tsf strains CBY9 (27) and BLY05 (this study), lacking PSHA, secreted significant levels of carboxypeptidase Y at the nonpermissive temperature (35 °C) (Fig. 1B). In contrast, the pep12–60tsf strain BLY05, expressing PSHA, secreted no more CPY than the wild-type PEP12 strain (CRY1), indicating a wild-type efficiency of CPY sorting (Fig. 1B). Loss of PEP12 function was lethal in the genetic background of JBY209 (data not shown), with the result that BLY05 failed to grow at 37 °C (Fig. 1C). PSHA expression in BLY05 restored growth at 37 °C (Fig. 1C). The complementation of loss of PEP12 function by PSHA demonstrates that PSHA is localized and functioning properly to mediate efficient delivery of CPY to the PVC.



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  		FIG. 1.
PVC-localized Kex2p substrate Pep12Ste13{Delta}TMD{alpha}HA provides functional Pep12p. A, schematic depiction of Pep12Ste13{Delta}TMD{alpha}HA (PSHA) fusion protein. Full-length Pep12p was fused to the luminal portion of Ste13p (Ste13{Delta}TMD, lacking cytosolic region and transmembrane domain). A Kex2p cleavage sequence (taken from pro-{alpha}-factor) and a triple hemagglutinin tag were fused to the C terminus of Ste13{Delta}TMD. B, PSHA expresses functional Pep12p. pep12–60tsf cells transformed with PSHA or control vector were analyzed for CPY secretion following 24 h of growth at 25 or 35 °C on nitrocellulose filter plates by immunoblot analysis of the filters with anti-CPY. C, PSHA restores growth at 37 °C to pep12tsf, ste13{Delta}, kex2{Delta}, pep4{Delta}, dap2{Delta} strain, BLY05. BLY05 and BLY05 pPSHA were analyzed for growth at 26 and 37 °C on yeast extract-peptone-dextrose medium + adenine plates.

 
Cleavage of PSHA by Kex2p in Vivo and in a Cell-free Reaction—The ability to measure transport between TGN membranes containing Kex2p and PVC membranes harboring PSHA depends on the ability of Kex2p to access and cleave the Kex2p-specific cleavage site separating Pep12-Ste13 (PS) from the HA tag (HA) of the PSHA chimera following co-integration of the two molecules into the same membrane compartment (Fig. 2A). Subsequent to transport and cleavage, the remaining intact PSHA can be immunoprecipitated by anti-HA antibody in the presence of nonionic detergent. The fraction of Ste13p dipeptidyl aminopeptidase activity remaining in the supernatant of the immunoprecipitation represents the fraction of PSHA cleaved by Kex2p and therefore the extent of transport.



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  		FIG. 2.
PSHA is processed by Kex2p in vivo and in vitro. A, schematic depiction of Kex2p transport from TGN to PSHA-containing PVC compartment. B, PSHA is processed by Kex2p in vivo. Membranes isolated from semi-intact spheroplasts of strain JBY209 transformed with either pPSHA alone (kex2{Delta}) or pPSHA and pKex2 (KEX2+) were assayed for the percentage of total DPAP activity that remains in the supernatant of an anti-HA immunoprecipitation in detergent. C, membranes isolated from semi-intact spheroplasts of strain JBY209 untransformed (kex2{Delta}) or transformed with pKex2 (KEX2+) were each combined with membranes isolated from JBY209 transformed with pPSHA in cell-free TGN-PVC transport assays.

 
To determine the accessibility of PSHA to Kex2p cleavage in vivo, PSHA was co-expressed with Kex2p in JBY209. Membranes isolated from this strain were subjected to anti-HA immunoprecipitation in the presence of 1% (v/v) Triton X-100 to determine the level of in vivo PSHA cleavage. As shown in Fig. 2B, ~40% of the PSHA was cleaved in vivo, and this cleavage was Kex2-dependent, demonstrating that a large fraction of the PSHA is accessible to Kex2 cleavage during normal cycling of Kex2p through the PVC.

To measure Kex2-dependent cleavage of PSHA in vitro, membranes prepared from a Kex2p-expressing strain lacking DPAP activity (ste13{Delta} dap2{Delta}) were combined with membranes prepared from a strain expressing PSHA but lacking Kex2p and all other sources of DPAP activity (kex2{Delta}ste13{Delta}dap2{Delta}). Following a 20-min incubation at 30 °C, ~10% of the PSHA was cleaved in a Kex2p-dependent fashion, as evidenced by the fraction of soluble DPAP activity measured (Fig. 2C). This level of cleavage represents the reconstitution of ~25% of the level of Kex2-dependent cleavage of PSHA observed in vivo under conditions of continual cycling of Kex2p (40%; Fig. 2B).

Cell-free Processing of PSHA by Kex2p is Temperature-, Time- and Energy-dependent—Cell-free membrane transport and fusion reactions typically require physiological temperatures and are ATP-dependent. Furthermore, reactions involving transport vesicle formation and fusion exhibit a pronounced lag. Cell-free processing of PSHA was maximal at 25–30 °C, consistent with optimal yeast growth temperatures (Fig. 3A), and proceeded with biphasic kinetics (Fig. 3B). An initial rapid phase (30% of total cleavage) was complete within 2.5 min. A second phase (70% of total) commenced after a 10-min lag and was complete after 20 min of incubation (Fig. 3B). Kex2p-dependent cleavage was largely dependent on the presence of an ATP regeneration system. Cleavage was nearly equivalent to background (kex2{Delta}) levels when phosphocreatine and creatine kinase were omitted from the reaction (Fig. 3C).



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  		FIG. 3.
PSHA processing is dependent on time, temperature, and ATP. A, TGN-to-PVC transport reactions between PSHA and KEX2+ or kex2{Delta} membranes were carried out at the indicated temperatures. B, TGN-to-PVC transport reactions between PSHA and KEX2+ or kex2{Delta} membranes were incubated at 30 °C for the indicated times. C, TGN-to-PVC transport reactions were carried out in the presence or absence of a phosphocreatine/creatine kinase ATP regeneration system.

 
Kex2p and PSHA DPAP Activity Are Co-localized in a Kex2p-immunoreactive Compartment following Transport—Vesicular transport of Kex2p to PVC membranes would be expected to result in co-integration of Kex2p and PSHA in the same lipid bilayer. To determine whether incubation of membranes under reaction conditions resulted in incorporation of the two proteins into the same membrane compartment, product membranes were immunoisolated using affinity-purified antibodies directed against the Kex2p cytosolic tail (33) by a method that results in quantitative isolation of membranes containing Kex2p (31). In reactions with Kex2p-containing membranes and the ATP regenerating system, ~12% of the PSHA was cleaved, whereas in reactions carried out using kex2{Delta} membranes or Kex2p-containing membranes in the absence of the ATP regenerating system, ≤4% of PSHA was cleaved (Fig. 4A). A portion of each reaction was subjected to immunoisolation on magnetic beads coated with anti-Kex2p cytosolic tail antibodies as described (25). Following immunoisolation, beads were assayed for Kex2p and DPAP activity. For both reactions having Kex2p-containing membranes, >90% of Kex2p activity was associated with the beads after immunoisolation (data not shown). Membranes immunoisolated from reactions having Kex2-containing membranes and the ATP regenerating system contained roughly 3% of the total DPAP activity (Fig. 4B). Membranes from reactions using kex2{Delta} membranes or lacking the ATP regenerating system contained only ~1% of the total DPAP (Fig. 4B). The percentage of DPAP activity recovered in each case was about 4-fold lower than the percentage of PSHA cleaved as determined by anti-HA immunoisolation. This is likely because of a partial loss of membrane integrity during the lengthy multiple high salt wash anti-Kex2p immunoisolation procedure versus the 30-min wash-free anti-HA immunoprecipitation carried out to determine the percentage of PSHA processed. Despite the loss in total signal for each reaction, the ratio of the amount of DPAP activity recovered from the standard reaction relative to that recovered from the control reactions corresponds well to the signal-to-noise ratio of the levels of PSHA processed between those same reactions (3 to 1). Thus, incubation of Kex2p and PSHA membranes results in ATP-dependent co-integration of Kex2p and PSHA in the same membrane.



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  		FIG. 4.
Kex2p and DPAP enzymatic activities are co-localized in a Kex2p-immunoreactive compartment following TGN-PVC transport. A, cell-free TGN-PVC transport reactions between KEX2+, kex2{Delta}, or KEX2+ without an ATP regeneration system (no regen) and PSHA-containing membranes were carried out. B, an aliquot of each reaction shown in A was incubated with magnetic beads coated with antibody to the cytosolic tail of Kex2p. Following several washes, beads were analyzed for Kex2p (data not shown) and DPAP activity.

 
Transport between Kex2p and PSHA Organelles Is Not Dependent on Vesicular Transport Out of the Late Endosome/Prevacuolar Compartment—As mentioned in the introduction, Kex2p maintains steady-state localization to the TGN by cycles of transport between the TGN and the PVC. As a result, Kex2p is actively transported both from the TGN to the PVC and from the PVC back to the TGN in living cells. It is therefore possible that cell-free, Kex2p-dependent cleavage of PSHA in this transport assay is the result of vesicular transport of Kex2p back to the TGN where it may encounter low levels of PSHA en route to the PVC as part of the biosynthetic pathway. One way to address the question of whether retrograde transport from the PVC to the TGN is contributing to Kex2p cleavage of PSHA is to examine the reaction using a class E VPS mutant. VPS27 encodes a member of the class E vacuolar-sorting proteins; their activity is required not only for multivesicular body formation at the PVC but also for transport out of the PVC both to the TGN and to the vacuole (26, 37). If PSHA cleavage depended on retrograde transport from the PVC to the TGN, a defect in the processing of PSHA would be observed in reactions utilizing membranes from vps27ts cells. As shown in Fig. 5, neither Kex2p membranes nor PSHA membranes prepared from vps27ts strains incubated at the nonpermissive temperature (35 °C) for 30 min prior to lysis exhibited significant defects in PSHA cleavage in the cell-free transport reaction. There was a slight decrease in the level of PSHA cleavage by vps27ts Kex2p membranes (~20%); however, this may have been due to slightly lower levels of Kex2p activity in the TGN following preincubation of vps27ts cells at the nonpermissive temperature because Kex2p activity accumulates in the PVC over time in class E VPS mutants (26, 38).



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  		FIG. 5.
Transport between Kex2p and PSHA organelles is not dependent on vesicular transport out of the late endosome/prevacuolar compartment. vps27ts membranes are competent for TGN-to-PVC transport. Kex2p and PSHA membranes were prepared from spheroplasts expressing a chromosomal temperature-sensitive allele of VPS27, incubated for 30 min. at 35 °C, and the function of these membranes was analyzed in a cell-free transport assay.

 
Transport between Kex2p and PSHA Organelles Requires Pep12p—We reported previously the cell-free reconstitution of homotypic TGN fusion. TGN fusion was found to require a t-SNARE complex consisting of Tlg2p, Tlg1p, and Vti1p (25). In contrast, cell-free delivery of Kex2p from the TGN to the PVC would be expected to require the t-SNARE Pep12p, which mediates the fusion of all incoming vesicular traffic at the PVC. To examine the function of Pep12p in TGN-to-PVC transport, two approaches were taken. First, F(ab) fragments of a monoclonal anti-Pep12p antibody were tested for inhibition of transport. Fig. 6A illustrates that the addition of anti-Pep12p F(ab) reduced transport by 90%. Second, the temperature-sensitive PEP12 allele, pep12–60tsf (27), was subcloned into the PSHA substrate construct replacing the wild-type PEP12 gene and generating PSHAtsf, a PVC-localized Kex2p substrate that was temperature-sensitive for Pep12p function as measured by temperature-sensitive CPY secretion (data not shown). The PSHAtsf-expressing construct was transformed into strain BLY05, isogenic with the strain used for fusion assays but containing genomic pep12–60tsf in place of wild-type PEP12. The resulting strain was grown at the permissive temperature to prevent perturbation of PVC trafficking in vivo. Membranes were prepared and preincubated at either 25 °C (permissive temperature) or 35 °C (nonpermissive temperature) for 10 min. Following preincubation at 25 °C, transport reactions containing PSHAtsf membranes exhibited reduced PSHA cleavage compared with reactions containing PSHA membranes (Fig. 6B). Preincubation at 35 °C resulted in a further reduction of transport in reactions containing PSHAtsf membranes but did not have a significant effect on reactions containing PSHA membranes (Fig. 6B). This is consistent with impaired function of the mutant pep12 allele in vitro that is exacerbated at the nonpermissive temperature. We conclude from these data that Pep12p function is required for the cell-free delivery of Kex2p into PSHA-containing compartments.



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  		FIG. 6.
TGN-to-PVC transport is dependent on Pep12p. A, anti-Pep12p F(ab) inhibits TGN-to-PVC transport. Anti-Pep12p was subjected to papain cleavage to create F(ab) fragments against the cytosolic domain of Pep12p. A mock papain cleavage reaction (Mock) was performed to control for potential papain-dependent inhibition of TGN-to-PVC transport. B, pep12tsf membranes are compromised for TGN-to-PVC transport. Membranes expressing a temperature-sensitive allele of PEP12, pep12–60tsf, both on the genome and as the Pep12p component of the PSHA substrate chimera (PSHAtsf) were preincubated at either 25 °C or 35 °C prior to initiation of transport reactions with Kex2p-membranes.

 
Transport between Kex2p and PSHA Organelles Does Not Require the TGN t-SNARE Tlg2p—As shown above, cell-free TGN-to-PVC transport required Pep12p function, unlike TGN homotypic fusion, which was found to be Pep12p-independent (25). The TGN-to-PVC transport reaction could be distinguished from homotypic TGN fusion by two additional criteria. Unlike TGN fusion, PSHA processing was completely resistant to IgG directed against the cytosolic domain of the TGN t-SNARE, Tlg2p (Fig. 7A). TGN homotypic fusion was recently found to be sensitive to inhibition by latrunculin B (data not shown). Whereas 0.5 mM latrunculin B inhibited TGN fusion by >60%, PSHA cleavage was entirely resistant to the drug (Fig. 7B). These experiments verify that cell-free TGN-to-PVC transport is biochemically distinct from homotypic TGN fusion. In addition to providing evidence that PSHA processing represents a new and distinct cell-free transport assay, these data also demonstrate that this transport event does not require either Tlg2p or actin.



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  		FIG. 7.
Transport between Kex2p and PSHA organelles is distinguishable from homotypic TGN fusion. A, TGN-PVC transport does not require Tlg2p. IgG (10 µg) purified from polyclonal anti-Tlg2p antisera was added to cell-free TGN-PVC transport and TGN fusion reactions. B, TGN-PVC transport is latrunculin-resistant. Latrunculin B (0.5 mM) or methanol (solvent) was added to cell-free TGN-PVC transport and TGN fusion reactions.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Protein sorting at the TGN is a complex process involving the formation of multiple distinct vesicular transport intermediates, each containing specific cargo proteins and each destined for delivery to a specific target compartment. In addition to the sorting of cargo passing transiently through the TGN, the cell must also maintain the TGN localization of transmembrane proteins such as Kex2p, Ste13p, and Vps10p. These proteins all achieve steady-state TGN localization by cycles of transport between the TGN and endosomes. Therefore, Kex2p, Ste13p, and Vps10p are also cargo molecules in the essential vacuolar/lysosomal biogenesis transport step between the TGN and the late endosome/prevacuolar compartment (3, 4). We have set out to characterize more thoroughly the mechanisms of transport between the TGN and endosomes by isolating distinct steps in transport of Kex2p between the TGN and endosomal membranes in cell-free systems. In this study, we have reconstituted Kex2p transport specifically from the TGN to the PVC.

Delivery of Kex2p to the PVC was rapid and resulted in the Kex2p-dependent cleavage of the PVC-localized Kex2p substrate, PSHA. Cell-free PSHA cleavage exhibited a biphasic time course. The two phases of this reaction may represent the initial fusion of preformed Kex2p-containing transport vesicles with the PVC followed by a second round of fusion resulting from the generation of additional Kex2p-containing transport vesicles during the reaction. Such preexisting transport vesicles may correspond to the clathrin-coated vesicles containing Kex2p that have recently been isolated from yeast lysates (9). Furthermore, the lag time for formation and delivery of transport vesicles in other cell-free transport systems (~10 min) corresponds well to the time of initiation of the second phase of the reaction (Fig. 3B) (21, 24, 30).

One concern of protease-substrate-mixing assays such as the one described here is that proteolytic cleavage of the substrate could occur independently of vesicle-mediated transport, for example, through a loss of organelle integrity that allows access between luminal contents of distinct organelle populations. The biochemical characteristics of this reaction strongly suggest that it represents an active process of vesicle-mediated transport. The reaction requires an ATP regenerating system. Although the source of the ATP requirement remains to be determined, likely candidates include the N-ethylmaleimide-sensitive factor (39, 40) and Hsc70p (41–43). However, PSHA cleavage was insensitive to N-ethylmaleimide (data not shown). Co-immunoisolation of DPAP activity provided independent confirmation that fusion between Kex2p- and PSHA-containing membranes occurs during the cell-free reaction.

Several transport steps other than TGN-to-PVC transport could hypothetically result in the processing of the PVC-localized substrate, PSHA, in the cell-free system. One possibility is that PSHA processing occurs by retrograde transport of PVC-localized Kex2p back to TGN compartments containing biosynthetic PSHA in transit through the Golgi. Strong evidence against a role for PVC-to-TGN transport in cell-free PSHA processing comes from analysis of vps27ts mutant membranes. As VPS27 function is required for vesicular transport out of the PVC (26, 37), the ability of vps27ts membranes to support Kex2p-dependent PSHA cleavage in the cell-free transport assay indicates that retrograde transport from the PVC to the TGN is not required. A second hypothetical possibility, that the reaction described here simply represents TGN homotypic fusion, was ruled out by the fact that the cell-free TGN-to-PVC transport reaction required Pep12p function and was insensitive to latrunculin B and to anti-Tlg2p antibody. A third possibility is that PSHA processing results from delivery of Kex2p from early to late endosomes. In addition to the cycles of transport between the TGN and the PVC, Kex2p has also been shown to be delivered to early endosomes (44) and to undergo transport from early endosomes to the PVC (45). A role for transport between early and late endosomes in PSHA cleavage is more difficult to exclude, in part because of the lack of definitive characterization of this transport step. In addition, distinguishing between the TGN and the early endosome as Kex2p donor compartment is challenging because of the similar biochemical characteristics and protein components of these two organelles. However, the conclusion that PSHA processing results from TGN-derived Kex2p vesicle fusion at the PVC is supported by the steady-state, and therefore predominant, localization of Kex2p at the TGN. In addition, we have found that replacing the Kex2p cytosolic tail with that of the CPY sorting receptor, Vps10p, results in cell-free PSHA processing equivalent to that achieved by wild-type Kex2p.2 Vps10p, unlike Kex2p, does not appear to traffic through early endosomes (14, 15, 45).

In conclusion, this study demonstrates the first cell-free assay in any system that reconstitutes transport from the TGN to the prevacuolar compartment/late endosome. We have used this assay to demonstrate directly a requirement for ATP and the syntaxin homolog Pep12p in delivery of a TGN resident protein Kex2p to the late endosome/PVC. This assay provides the means to pursue the mechanistic dissection of this critical sorting step in vacuolar/lysosomal biogenesis and, more specifically, to elucidate the sequence of events that lead to transport vesicle formation and cargo recruitment at the TGN followed by targeted delivery and vesicle fusion at the PVC.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants GM50915 and GM39697 (to R. S. F.), a University of Michigan Rackham Graduate School predoctoral fellowship (to J. M. B.), a University of Michigan Medical Scientist Training Program grant (to M. E. A.), Genetics Training Program Grant GM07544 (to J. M. B. and M. E. A.), and National Cancer Institute Grant 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} These authors contributed equally to this work. Back

§ To whom correspondence should be addressed: Dept. of Biological Chemistry, University of Michigan, 1301 Catherine Rd., 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; PVC, prevacuolar compartment; CPY, carboxypeptidase Y; t-SNARE, target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor; HA, hemagglutinin; PSHA, Pep12Ste13{Delta}TMD{alpha}HA; IP, immunoprecipitation or immunoprecipitate; DPAP, dipeptidyl aminopeptidase; VPS, vacuolar protein sorting. Back

2 M. E. Abazeed, J. M. Blanchette, and R. S. Fuller, submitted for publication. Back


   ACKNOWLEDGMENTS
 
We are grateful to Scott D. Emr and Hugh R. B. Pelham for generously providing strains and plasmids and to members of the Fuller laboratory for helpful comments on the manuscript.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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