Report |
Address correspondence to David E. James, The Garvan Institute of Medical Research, 384 Victoria St., Darlinghurst, Sydney, NSW, Australia 2010. Tel.: 61-2-9295-8210. Fax: 61-2-9295-8201. E-mail: d.james{at}garvan.org.au
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Abstract |
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Key Words: SNAREs; fusion; protein phosphatase 1; docking; membranes
* Abbreviations used in this paper: CPY, carboxypeptidase Y; HA, hemagglutinin; PP1, protein phosphatase 1; SM, Sec1p/Munc18.
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Introduction |
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SNARE complex assembly occurs in several discrete stages. Initially, in a priming step, cis-SNARE complexes in both donor and target membranes are disassembled through the ATPase action of NSF in conjunction with its attachment protein -SNAP (Mayer et al., 1996). Once primed, tethering machinery may guide the SNAREs in each membrane into close proximity and a proofreading machinery likely ensures fidelity of the aligned v- and t-SNARE pairs (Zerial and McBride, 2001). Formation of the high affinity trans-SNARE complex represents the committed step of docking (Ungermann et al., 1998). This step is accompanied by conversion of trans- to cis-complexes (Lin and Scheller, 1997; Sutton et al., 1998). Although SNARE proteins are sufficient to catalyze docking and fusion of artificial membrane liposomes in vitro (Weber et al., 1998; McNew et al., 2000), other factors are essential in vivo, suggesting multiple levels of regulation.
The Sec1p/Munc18 (SM)* family plays an essential role in regulating membrane transport (Jahn, 2000). Disruption or deletion of any of the four SM proteins in Saccharomyces cerevisiae causes a block in vesicle transport (Jahn, 2000). SM proteins are peripheral membrane proteins. We have demonstrated previously that the SM protein Vps45p is dependent on its cognate t-SNARE Tlg2p for its membrane association (Bryant and James, 2001). Moreover, it has been shown that many SM proteins bind their cognate t-SNARE with high affinity in vitro (Pevsner et al., 1994b; Yamaguchi et al., 2002), suggesting that the stable association of SM proteins with membranes is mediated via this interaction. But how this interaction changes as the t-SNARE engages in core complex assembly during docking is not known. At the neuronal synapse, it is thought that the SM protein Munc18a is inhibitory to core complex assembly and that its dissociation from the t-SNARE is required to promote this process (Pevsner et al., 1994a). Neither the fate of the liberated SM protein, or the mechanism that underlies its dissociation is known. One possibility is that SM proteins dissociate from membranes before core assembly. Intriguingly, many SM proteins, including Vps45p are present as both cytosolic and membrane-bound forms (Cowles et al., 1994; Bryant and James, 2001). The cytosolic pool may represent newly synthesized protein or Vps45p that has been discharged from the membrane during vesicle transport. In either case, it is not clear at what stage SM proteins achieve their membrane association. Intriguingly, Sec1p is found stably associated with the cis-SNARE complex (Carr et al., 1999), suggesting that the terminal stage of the transport reaction may be the point at which SM proteins reassociate with the membrane in order to reactivate the t-SNARE for another round of vesicle transport.
Here, we have found that the SM protein Vps45p dissociates from membranes before fusion and reassociates after fusion probably by binding to a cis-SNARE complex. The cyclical membrane association of Vps45p is temporally linked to the dockingfusion stage of vesicle transport at a step controlled by the protein phosphatase Glc7p. We propose that the dissociation of SM proteins from the membrane may be controlled by phosphorylation and that this family of proteins may act as molecular switches to control SNARE complex assembly.
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Results and discussion |
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Fig. 3 demonstrates that there is a significant three- to fivefold increase in the amount of Tlg2p, Vti1p, and Snc2p that coprecipitate with Tlg1p when cells harboring a temperature-sensitive allele of GLC7 are incubated at the nonpermissive temperature (Fig. 3 A). Intriguingly, no Vps45p was found associated with the SNARE complexes that accumulate in these cells (Fig. 3 A, 37°C). This is in contrast to the cis-SNARE complexes that accumulate in sec181 cells (Fig. 1). The observation that Vps45p binds to SNARE complexes present in wild-type cells, and also to those detected in sec181 cells at the permissive temperature, indicates that these are cis-SNARE complexes, whereas those that accumulate in glc710 cells at 37°C are in the trans-configuration.
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To assess the physiological relevance of the loss of membrane association upon inactivation of PP1, we sought to ascertain whether the dissociation observed upon inactivation of PP1 was reversible upon reactivation of the phosphatase. Fig. 4 A demonstrates that the block in membrane trafficking caused by incubation of glc710 cells is reversible. It has been shown previously that the transit of the vacuolar hydrolase carboxypeptidase Y (CPY) through the secretory pathway is blocked in PP1 temperature-sensitive cells at 37°C (Peters et al., 1999). After its synthesis, CPY is translocated into the lumen of the ER, where it is core glycosylated into the p1 form. CPY receives further modifications as it transits through the Golgi apparatus. In the trans-Golgi network, the p2 form of CPY is recognized by its receptor Vps10p, which delivers the protein to the vacuole where it is cleaved into the mature form of the protease (Bryant and Stevens, 1998). Fig. 4 A demonstrates that incubation of glc710 cells at 37°C blocks the processing of CPY. Concomitant with this block in vesicle fusion is the dissociation of Vps45p from membranes, as demonstrated by the observation that the SM protein shifts from the P100 (membrane) to the S100 (cytosol) fraction obtained from these cells after 10 min at 37°C (Fig. 4 B). The block in processing of CPY was reversed by returning the cells to 24°C (Fig. 4 A). Importantly, with this recovery in vesicular transport, Vps45p reassociated with membranes (Fig. 4 B). These data indicate that the redistribution of Vps45p from membranes to the cytosol, observed in glc710 cells (Figs. 3 and 4) represents a physiological intermediate.
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In a manner similar to the finding that Sec1p binds to SNARE complexes (Carr et al., 1999), we observed a robust interaction between Vps45p and Tlg2p in sec181 cells (Fig. 1). It is noteworthy that in a previous study, Lupashin and Waters (1997) reported that there was no significant interaction between Sly1p- and Sed5p-containing SNARE complexes that accumulate in sec181 cells, despite the fact that Sly1p has subsequently been shown to interact with Sed5p-containing SNARE complexes formed in vitro (Kosodo et al., 2002; Peng and Gallwitz, 2002). However, in that study (Lupashin and Waters, 1997), sec181 mutants were incubated for times significantly longer than those used here, and we have found that the period of incubation of these cells at the nonpermissive temperature is critical in these experiments (unpublished data).
Our model raises a number of important questions that provide a framework for further investigation directed at understanding the role of SM proteins. Why does Vps45p dissociate from the membrane during the dockingfusion stage? Do SM proteins play a role, similar to Rab proteins, and cycle on and off membranes in synchrony with the arrival of vesicles at the docking site? In addition, our paper raises questions concerning the role of PP1 in dockingfusion events. What is the role of phosphorylation that appears to be so intimately linked to the dockingfusion transition of vesicle transport; and is the dissociation of the SM protein during this phase the cause, or an effect, of this? Intriguingly, a role for the phosphorylation of Tlg2p has been implicated in modulating endocytosis (Gurunathan et al., 2002), and phosphorylation of Munc18a may be important in regulating its interaction with Syntaxin1a (Fujita et al., 1996). Finally, with our finding that Vps45p binds selectively to certain SNARE intermediates, could it be that there is a consensus in SM function that has been thus far difficult to realize? If phosphorylation plays an important role in the SMSNARE interaction, replicating the conditions necessary to establish these interactions may not be trivial. Notably, Carr et al. (1999) stressed the importance of eliminating ATP from their cells before isolation of intact SNARE complexes. Answers to the above, and other, questions will further our understanding of this important family of proteins.
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Materials and methods |
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Plasmids
All DNA manipulations were performed using routine procedures. Plasmid pEG111, provided by E. Grote (Yale University, New Haven, CT) is an integrating vector encoding an HA-epitopetagged version of Snc2p (Abeliovich et al., 1998). pNOz10 was constructed by subcloning a BamHI-HindII fragment encompassing the tagged Snc2p coding sequence from pEG111 into pRS306 (Sikorski and Hieter, 1989).
Strains
All yeast strains used in these studies are described in Table I. Yeast strains were constructed using standard genetic techniques and grown in rich media (YEPD; 1% yeast extract, 1% peptone, 2% dextrose) or standard minimal medium using either glucose, or raffinose and galactose as carbon sources. NOzY21, 22, 23, and 24 all harbor an allele of SNC2 encoding an HA-tagged version of the v-SNARE (Abeliovich et al., 1998). NOzY21, NOzY23, and NOzY24 were derived from SF8389D (Rothman and Stevens, 1986), PAY7041, provided by M. Stark (University of Dundee, Dundee, UK; Peters et al., 1999), and SGY79 (Gerrard et al., 2000), respectively, using pEGIII (Abeliovich et al., 1998). NOzY22 was constructed from SEY5186, provided by S. Emr (University of California, San Diego, La Jolla, CA), using pNOz10.
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SNARE complex immunoprecipitations
Tlg1p- and Tlg2p-containing complexes were immunoprecipitated from solubilized membranes as described previously (Abeliovich et al., 1998; Bryant and James, 2001). Precipitated proteins were detected using immunoblot analysis. Band intensities were quantified by densitometry using a scanning densitometer (Bio-Rad Laboratories) and NIH Image software.
Metabolic labeling and pulse-chase immunoprecipitation of CPY
The fate of newly synthesized CPY in glc710 cells was followed by immunoprecipitation of the protein as described previously (Bryant and James, 2001).
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Acknowledgments |
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This research was supported by the National Health and Medical Research Council of Australia.
Submitted: 13 December 2002
Revised: 4 April 2003
Accepted: 8 April 2003
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