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Address correspondence to Robert S. Fuller, Dept. of Biological Chemistry, Rm. 5413, MSI, 1301 Catherine Rd., University of Michigan Medical School, Ann Arbor, MI 48109-0606. Tel.: (734) 936-9764. Fax: (734) 763-7799. E-mail: bfuller{at}umich.edu
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Abstract |
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Key Words: TGN; rab; SNARE; fusion; Kex2p
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Introduction |
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The trans-Golgi network (TGN) consists of a complex network of vesicles and tubules in continual communication with Golgi cisternae, early and late endosomes, and the cell surface. Numerous sorting, budding, and fusion events must occur in a coordinated fashion to maintain the functional integrity of the TGN and prevent undesirable mixing of endocytic and secretory cargo. The molecular details of how this is achieved are unclear. One approach toward understanding sorting in this organelle is to reconstitute TGN membrane fusion events.
We have developed an assay to monitor vesicular transport and membrane fusion events between the TGN and endosomal compartments in yeast. Using this assay, we have discovered a vigorous and previously uncharacterized membrane fusion event involving organelles containing the TGN resident enzymes Kex2p and Ste13p (dipeptidyl aminopeptidase [DPAP]A). Membrane fusion was energy and cytosol dependent. It was inhibited by rapid chelation of Ca2+ and the thiol-reactive reagent NEM.
Furthermore, the SNARE proteins Tlg1p, Tlg2p, and Vti1p, the rab5 homologue Vps21p, and the Sec1p homologue Vps45p, all of which have been implicated in late Golgi/endosomal function in vivo, are required for this reaction. NSF-sensitive SNARE complexes containing various combinations of Tlg1p, Tlg2p, and Vti1p have been isolated (Holthuis et al., 1998; Nichols et al., 1998; Coe et al., 1999). TGN homotypic fusion represents an event in which these three SNAREs appear to function together.
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Results |
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Discussion |
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Mutations in the genes encoding the components of the Tlg SNARE complex give rise to distinguishable phenotypes, suggesting that each of these proteins catalyzes an unique set of fusion events (von Mollard et al., 1997; Abeliovich et al., 1999; Coe et al., 1999). The fusion event we measure here appears to represent one reaction in which these three proteins participate together. Consistent with our data, a t-SNARE complex of Tlg1p, Tlg2p, and Vti1p has been found to pair specifically with Snc1p to catalyze fusion when reconstituted in liposomes (Paumet et al., 2001). Preliminary evidence suggests that Snc1p is likely to function as the v-SNARE in conjunction with the Tlg SNARE complex in homotypic TGN fusion (unpublished data); however, this remains to be conclusively demonstrated. This study provides a biochemical assay for the coordinate function of all of these proteins in a single fusion event involving physiological membranes.
Steady-state localization of transmembrane proteins to the TGN in yeast depends on continuous cycles of transport between TGN and post-Golgi endosomal compartments (Vida et al., 1993; Cereghino et al., 1995; Piper et al., 1995; Cooper and Stevens, 1996; Brickner and Fuller, 1997; Bryant and Stevens, 1997). Thus, this reaction could hypothetically represent vesicular transport events between the TGN and the PVC. However, our data suggest that the reaction described here does not correspond to a vesicular transport event. First, the reaction did not display the extended lag phase typical of cell-free vesicular transport (Balch et al., 1984; Baker et al., 1988; Vida and Gerhardt, 1999). Second, whereas the PVC t-SNARE Pep12p is required for TGN to PVC transport and the class E Vps protein Vps27p is required for PVC to TGN transport, the reaction we observed required neither protein based on the following observations. Membranes prepared from a pep12 temperature-sensitive mutant strain (Burd et al., 1997) were able to support processing of Ste13HA after a 5-min preincubation at the restrictive temperature to inactivate the protein (unpublished data). Moreover, anti-Pep12p antiserum failed to inhibit the reaction. Membranes prepared from vps27-null or temperature-sensitive mutants were fully competent to support processing of Ste13
HA (unpublished data). Finally, there was no requirement for the trans-Golgi localization signal (TLS)1 (Brickner and Fuller, 1997) in the cytosolic tail of Kex2p, which would be required if the reaction involved transport of Kex2p from the PVC to the TGN (unpublished data).
Several lines of evidence argue that the reaction described here measures a homotypic fusion event. Homotypic fusion involves fusion of like organelles through the pairing of t-SNAREs and v-SNAREs distributed comparably onto the fusing membranes. In the case of homotypic vacuolar fusion, v-SNAREs can be genetically ablated from one membrane and t-SNAREs from the other and fusion can still proceed (Wickner and Haas, 2000). In contrast, heterotypic fusion requires pairing of a v-SNARE on a vesicle with t-SNAREs on the target membrane. The absence of either blocks the reaction. Ideally, mutations of SNARE genes therefore can allow the classification of a fusion reaction as homotypic or heterotypic as in the case of vacuolar fusion (Wickner and Haas, 2000). However, because the existence of the compartments to which our reporter proteins are localized depends on the components of the Tlg SNARE complex, such an experiment would be difficult to interpret. An alternative way to distinguish homotypic and heterotypic fusion is the sensitivity of the reaction to pretreatment of one of the membranes with antiserum against one of the participating SNARE proteins. We have found that in the reconstituted reaction involving pelleted membranes and cytosol pretreatment of either membrane before centrifugation with antisera against Tlg1p or Tlg2p was sufficient to completely inhibit the fusion reaction (unpublished data). Although this result is somewhat different from what was found in vacuolar fusion (see above), it argues that identical v- and t-SNAREs are present and are functional in the reaction on both of the fusing membranes, a necessary characteristic of homotypic fusion. Vps21p rab function was required, but on only one of the two membranes, to support membrane fusion. The symmetry inherent in the fact that rab activity on either membrane was sufficient supports the conclusion that the reaction occurs between two identical membranes.
Because both Kex2p and Ste13p cycle between the TGN and late endosomal compartments and possibly through early endosomal compartments (unpublished data) the fusion reaction between Kex2p- and Ste13HA-containing membranes could represent homotypic fusion of TGN or early or late endosomal membranes. It seems unlikely that this reaction involves late endosomal/PVC membranes because (a) antibodies against Pep12p, the major late endosomal t-SNARE, did not inhibit the fusion reaction and (b) MSS membranes from vps27-null mutants, which accumulate aberrant late endosomal membranes, exhibited normal fusion activity (unpublished data). Subsets of components of the Tlg SNARE complex have been implicated in endocytosis (Tlg1p, Tlg2p, and Vps21p [Abeliovich et al., 1998; Seron et al., 1998; Gerrard et al., 2000]), biosynthetic traffic to the vacuole (Vti1p and Vps21p [Horazdovsky et al., 1994; von Mollard et al., 1997]), and early endosome homotypic fusion in mammalian cells (rab5/Vps21p; [Gorvel et al., 1991]). These observations could implicate the involvement of early endosomal membranes in this reaction. Although this possibility cannot be excluded at this time, we believe it is more likely that the homotypic fusion reaction described here represents fusion of TGN membranes. First, up to 45% of Ste13
HA was processed in this cell-free reaction, suggesting that at least half of the Ste13
HA is localized to the membrane compartment that participates in the reaction. Ste13p has been shown to possess a strong TGN retention signal that slows its delivery to the late endosome, presumably by retarding its exit from the TGN (Bryant and Stevens, 1997). Therefore, it is unlikely that the majority of Ste13
HA is localized to the endocytic compartments. Second, Tlg1p and Tlg2p have been colocalized with Kex2p in the late Golgi and have been shown to be required for its proper localization and activity (Holthuis et al., 1998). These SNAREs do not colocalize extensively with Pep12p, a late endosomal marker, or with Sec7p, an early Golgi marker (Lewis et al., 2000).
We hope to use the cell-free fusion assay described here to analyze the function of the Tlg SNARE complex and identify the cytosolic components required for the reaction. The basic assay we have described may also be applied to reconstitute other transport and fusion events in the TGN/endosomal system by exploiting the use of distinct localization signals or accumulation in specific compartments to achieve differential localization of the enzyme (Kex2p) and substrate (Ste13HA). Finally, these results call attention to the need for developing assays for TGN homotypic fusion in vivo in order to understand its importance for TGN membrane protein localization and TGN function.
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Materials and methods |
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FCS was from GIBCO BRL. Boc-Leu-Lys-Arg-7-amino-4-methylcoumarin (LKR-AMC) was from Bachem and Ala-Pro-AMC (AP-AMC) was from Enzyme Systems Products. Restriction endonucleases and DNA modification enzymes were from New England Biolabs, Inc. Unless indicated otherwise, all other chemicals and reagents were from Sigma-Aldrich.
Strains and plasmids
The following strains were used: JBY209 (ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 kex2::hisG dap2
::kanr pep4
::HIS3 ste13
::LEU2 MAT
), JBY209a (isogenic with JBY209, except that it is MATa), and BLY9 (his3 leu2 trp1 ura3 kex2
::hisG dap2
::kanr pep4
::HIS3 ste13
::LEU2 vps21). BLY9 was constructed by crossing the vps21 allele from strain 0587-21 (provided by Dr. Scott Emr, University of California, San Diego, CA) into JBY209a and isolating His+, Leu+, G418r, Vps-, and kex2
segregants after sporulation. To create Kex2p- or Ste13
HA-expressing strains, plasmids pCWKX10 or pSTE13
HA, respectively, were introduced by transformation. Untransformed JBY209 was used as the kex2
control.
Plasmids pCWKX10 (Wilcox et al., 1992), containing the KEX2 gene under its own promoter, pTLG2HISN1 and pTLG1HISC1 (Holthuis et al., 1998), and pET28aHis6VTI1TMD (Fukuda et al., 2000) have been described. Plasmid pSTE13
HA was constructed as follows. The STE13 gene was amplified by PCR from pBRSTE13 (Jeremy Thorner, University of California, Berkeley, CA) using the following primers (5' to 3'): GTTATTCGTGTAAAAAATCTAGAAAGCCCCTA, which anneals directly upstream of the start codon and introduces an XbaI site and CAATCATCCATAAAGAATTCTAAATGCAAA, which anneals at the end of STE13 and both introduces an EcoRI site and changes the termination codon to GAA. The PCR product was ligated between the XbaI and EcoRI sites of p416TEF (Mumberg et al., 1995). The resulting plasmid, pTEF-STE13, was digested with EcoRI and SalI and ligated to the following phosphorylated oligonucleotides, which had been annealed to each other (5' to 3'): AATTGGATGCATCGGAATTCAGCGGCCGCTTG and TCGACAAGCGGCCGCTGAATTCCGATGCATCC. The resulting plasmid, pTEF-STE13 linker, was digested with NsiI and ligated to the following phosphorylated and annealed oligonucleotides (5' to 3'): TGGCATTGGTTGCAACTAAAACCTGGCCAACCAATGTACAAGAGAGATGCA and TCTCTCTTGTACATTGGTTGGCCAGGTTTTAGTTGCAACCAATGCCATGCA, encoding the following amino acid sequence from prepro
-factor: Trp His Leu Gln Leu Lys Pro Gly Gln Pro Met Tyr Lys Arg Asp Ala, confirmed by DNA sequencing. The resulting plasmid, pSTE13
, was digested with NotI and ligated to NotI-digested triple HA tag (Tyers et al., 1992), giving pSTE13
HA.
Preparation of membranes and cytosol
Permeabilized cells were prepared from JBY209 containing pCWKX10 or pSTE13HA as described (Baker et al., 1988). Frozen spheroplasts (200 µl) were thawed (25°C, 2 min) and centrifuged (5 min, 14,000 rpm), and MSS fraction (containing microsomes and cytosol) was collected. For assays in which membranes were separated from cytosol, MSS membranes were diluted fourfold with lysis buffer and centrifuged through a 250-µl band of 12.5% ficoll in a TLS55 rotor (Beckman Coulter) at 55,000 rpm (200,000 g at ravg) for 1 h at 4°C to generate a high speed pellet fraction (P200). The P200 was resuspended in lysis buffer to 15% of the MSS volume and assayed for Kex2p or DPAP activity. P200 fractions were diluted to the same specific activity as the MSS membranes; 10 µl were used per reaction. Cytosol was prepared by centrifugation of MSS from JBY209 in a TLS55 rotor at 55,000 rpm for 1 h at 4°C and was concentrated on microcon-10 columns (Millipore) to a final protein concentration of 2025 mg/ml as determined by the Bradford assay (Bio-Rad Laboratories).
Cell-free fusion
10 µl of MSS membranes from both the Kex2p- and Ste13HA-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, 0.375 mg ml-1 creatine kinase, and 9 mM CaCl2) 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 (IP) mix (1% Triton X-100, 1 mM EDTA, 20 µl pansorbin, 1 µl 12CA5 monoclonal anti-HA, and 1 µl rabbit antimouse IgG) or mock IP mix (1% Triton X-100, 1 mM EDTA, 20 µl pansorbin, and 2 µl water). IPs were incubated at RT with gentle agitation for 30 min. Pansorbin was pelleted, and 30 µl of each supernatant fraction were assayed for DPAP activity. The fraction of Ste13
HA 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 IP reaction (i.e., the total). Error bars represent the standard deviation of the average of at least two reactions.
Enzymatic assays
DPAP assays were performed as described (Julius et al., 1983). The supernatant fraction from the IP reactions (30 µl) was added to 30 µl 2x DPAP reaction mix (240 mM Hepes, pH 8, 1% Triton X-100, 200 µM AP-AMC), and reactions were incubated in 96-well plates at 30°C in an fmax plate fluorimeter (360 nm excitation/460 nm emission filter pair; Molecular Devices). Data were collected approximately every 20 s for 20 min. Kex2p was assayed as described (Brenner and Fuller, 1992) except that LKR-AMC was used as substrate and 5 mM o-phenanthroline was included to inhibit a membrane-associated Kex2p-independent activity present in crude lysates (Sipos and Fuller, 2001).
Immunoisolation of Kex2p membranes
Anti-Kex2p cytosolic tail antibodies were affinity purified (Redding et al., 1991). Dynabeads (Dynal) were covalently coated with affinity purified goat anti-rabbit IgG/Fc fragment-specific antibodies and bound to affinity purified anti-Kex2p tail antibodies as recommended by the manufacturer. Beads were washed three times with 50 mM Hepes, pH 7.0, 200 mM KOAc, 2 mM EDTA, and 5% FCS before use.
Reactions (scaled up twofold) were performed as described above. In control reactions, Kex2p- and Ste13HA-expressing membranes were incubated separately in reaction mix and combined after incubation. At the end of the incubation, half of each reaction was processed for IP or mock IP to determine processing efficiency. The remainder (30 µl) was adjusted to 50 mM Hepes, pH 7.0, 200 mM KOAc, 2 mM EDTA, 5% FCS, and 0.8 M sorbitol in a final volume of 50 µl and added to 150 µg Dynabeads. Binding was performed for 2 h with slow rotation at RT. Beads were isolated magnetically, washed three times with cold 50 mM Hepes, pH 7, 200 mM KOAc, and 0.8 M sorbitol and assayed for Kex2p and DPAP activity.
Preparation of soluble domains of Tlg2p, Tlg1 p, and Vti1p
His6-Tlg2TMDp was expressed in E. coli BL21 (DE3) and purified using ProBondTM (Invitrogen) according to manufacturer's denaturing protocol. Recombinant protein was eluted with 20 mM NaPO4, pH 4.0, and 1 M urea. Fractions enriched for recombinant protein were pooled, dialyzed in 20 mM NaPO4, pH 7.0, 1 M urea, and concentrated to 1020 mg ml-1 on microcon-10 columns. Tlg1-His6
TMDp and His6-Vti1
TMDp were expressed and purified as described above except that denaturants were omitted during lysis and purification and elution was performed with 400 mM imidazole-HCl in 20 mM NaPO4, pH 7.4, and 500 mM NaCl.
Antigen competition
Polyclonal antiserum (100 µg total protein) was incubated with 1040 µg recombinant protein on ice for 1 h. Antigen-saturated serum was then incubated with MSS membranes on ice for 1 h, and the membranes were tested for fusion competence.
IgG purification
Antisera were dialyzed overnight in 500-vol 20 mM KPO4, pH 7.2. Dialyzed serum was loaded onto a column containing equal volumes of CM- and DEAE-Sepharose. Flow through was collected and concentrated on microcon-10 columns. IgG purity and concentration were determined by SDS-PAGE and the Bradford assay.
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Footnotes |
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J.H. Brickner's present address is Dept. of Biochemistry and Biophysics, University of California, San Francisco, CA 94143.
* Abbreviations used in this paper: DPAP, dipeptidyl aminopeptidase; HA, hemagglutinin; IP, immunoprecipitation; MSS, medium speed supernatant; NEM, N-ethylmaleimide; NSF, NEM-sensitive fusion protein; P200, 200,000 g pellet; PVC, prevacuolar compartment; SNARE, soluble NSF attachment protein receptor; TGN, trans-Golgi network; TLS, trans-Golgi localization signal; TMD, transmembrane domain; t-SNARE, target membrane SNARE; v-SNARE, vesicle SNARE.
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Acknowledgments |
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This work was supported in part by National Institutes of Health grant GM50915 to R.S. Fuller and by Genetics Training grant GM07544 to J.M. Blanchette.
Submitted: 23 April 2001
Revised: 23 October 2001
Accepted: 23 October 2001
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