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Address correspondence to William Wickner, Dept. of Biochemistry, Dartmouth Medical School, 7200 Vail Bldg., Hanover, NH 03755-3844. Tel.: (603) 650-1701. Fax: (603) 650-1353
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Abstract |
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Key Words: yeast vacuoles; membrane fusion; actin; latrunculin B; jasplakinolide
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Introduction |
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Actin has a central role in several trafficking events (Qualmann et al., 2000; Foti et al., 2001; Goode and Rodal, 2001; Lechler et al., 2001). Actin filaments are required for the transport and spatial targeting of secretory proteins (Pruyne et al., 1998; Guo et al., 2001), organelle inheritance during cell division (Catlett and Weisman, 2000), and the maintenance of Golgi structure (Mullholland et al., 1997; Valderrama et al., 1998). In different systems, membrane fusion has been shown to be promoted by stabilization of F-actin (Koffer et al., 1990; Jahraus et al., 2001), F-actin disassembly (Vitale et al., 1991; Muallem et al., 1995), or actin remodeling, i.e., disassembly plus reassembly (Bernstein et al., 1998; Lang et al., 2000). Actin remodeling also accompanies synaptic stimulation (Colicos et al., 2001). Actin ligands can modulate phagosomeendosome fusion in vitro (Jahraus et al., 2001), and actin binds to purified endosomal and lysosomal vesicles, an association which can nucleate actin polymerization (Mehrabian et al., 1984; Taunton, 2001). Actin and myosin V are involved directly in vacuole movement into the bud during cell division (Hill et al., 1996; Catlett et al., 2000). Rho-GTPases regulate actin rearrangement (Hall, 1998) by signaling to multiple downstream effector complexes such as the Wiskott-Aldrich syndrome protein (WASp)* and the Arp2/3 complex (Higgs and Pollard, 1999). In each of these studies, it was assumed that the relevant actin molecules are cytosolic or cytoskeletal, though none of the data precludes a role for organelle-bound actin.
We study membrane fusion with vacuoles from Saccharomyces cerevisiae (Wickner and Haas, 2000). Purified yeast vacuoles undergo homotypic fusion in simple buffers containing ATP. All of the proteins and lipids needed for fusion are bound to the vacuole membrane. The reaction occurs in three stages termed priming, docking, and fusion. Priming, initiated by the ATPase Sec18p, releases Sec17p (Mayer et al., 1996) and disassembles a cis complex of SNAREs (Ungermann et al., 1998a). Priming liberates the "HOPS" complex (for homotypic fusion and vacuole protein sorting)/VPS class C complex (Sato et al., 2000; Seals et al., 2000), which then associates with GTP-bound Ypt7p to initiate docking (Price et al., 2000). Completion of docking requires SNAREs (Ungermann et al., 1998b), the vacuole membrane potential (Ungermann et al., 1999), phosphoinositides (Mayer et al., 2000), and the Rho-GTPases Cdc42p and Rho1p (Eitzen et al., 2001; Müller et al., 2001). Docking culminates in a transient release of vacuole lumenal calcium (Peters and Mayer, 1998). Calcium activates calmodulin, which binds to the V0 domain of the vacuolar ATPase, triggering the formation of trans-pairs of V0 plus the t-SNARE Vam3p, leading to organelle fusion (Peters et al., 2001).
Two Rho-GTPases which are required for vacuole fusion, Cdc42p and Rho1p (Eitzen et al., 2001; Müller et al., 2001), can regulate actin structure (Pringle et al., 1995; Helliwell et al., 1998) through a well-studied cascade which includes Las17p/Bee1p (yeast WASp) and the Arp2/3 complex (Fig. 1) . A recent screen of a library of yeast strains with defined gene deletions (Seeley et al., 2002) suggested that this cascade of actin regulatory genes is needed to maintain normal vacuole structure. We now report that the proteins of this regulatory cascade, from Cdc42p to Las17p and Arp2/3p, and actin itself, are found on purified yeast vacuoles, are essential for fusion, and allow actin action at the final stage of the fusion pathway. This role of actin in vacuole fusion may extend to other membrane fusion events.
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Results |
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The sensitivity of vacuole fusion to antibody to an NH2-terminal region of Las17p is partially suppressed by the addition of pure recombinant WCA domain of Las17p (Fig. 3 C). The COOH-terminal WCA domain of Las17p stimulates the Arp2/3 complex in yeast and higher eukaryotes (Machesky et al., 1999; Winter et al., 1999). We also found that addition of the CA domain partially suppresses anti-Las17p inhibition, although less efficiently than the WCA domain (Fig. 3 C). Whereas the CA domain of WASp acts as a dominant-negative inhibitor in mammalian extracts (Machesky et al., 1999), the CA domain of yeast stimulates Arp2/3 function, though less efficiently than WCA (Winter et al., 1999). The reaction sensitivity to anti-Las17p is also suppressed by the addition of calmodulin (Fig. 3 C), in accord with the finding that calmodulin activates the Arp2/3 complex, which is a downstream effector of Las17p/WASp (Schaerer-Brodbeck and Riezman, 2000a). Thus, actin remodeling may be at least one of the functions of calmodulin in vacuole fusion. Calmodulin also regulates many other cellular processes and specifically associates with the vacuolar Vtc complex (Müller et al., 2002); further studies will be needed to establish its full range of functions in vacuole fusion. We also used commercially available Arp3p antibodies to directly test the requirement of the Arp2/3 complex in our reaction. An antibody to an NH2-terminal domain of Arp3p antibody inhibits vacuole fusion, whereas antibody to the COOH-terminal domain of Arp3 or heat-denatured antibodies do not inhibit (Fig. 3 D). These biochemical studies complement the genetic results (Fig. 2 A) and indicate that the pathway from Cdc42p through Las17p to the Arp2/3 complex regulates vacuole fusion. Therefore, we sought direct tests of the role of actin itself in vacuole fusion.
Actin mutations affect vacuole fusion
Several actin mutants were examined for their vacuole morphology in vivo. Strains bearing the act1101 allele have fragmented vacuoles, whereas strains bearing the act1157 allele have abnormally large, lobed vacuoles (Fig. 2 A). Purified vacuoles from these strains, when incubated in vitro without added cytosolic proteins, are strikingly defective for in vitro fusion when compared with their isogenic ACT1 counterparts (Fig. 4
A, lanes 13). Though we have not found conditions which allow fusion of vacuoles from the act1101 strain, fusion is restored to act1157 vacuoles when given cytosol (lane 17) or a mixture of the purified chaperone IB2 (Slusarewicz et al., 1997), calmodulin, and Sec18p (lane 14), a mixture which we term "pure components" (P.C.). This agrees well with the failure of in vivo fusion of act1101 vacuoles but a fused, albeit abnormal, structure of act1157 vacuoles (Fig. 2 A). These P.C. which stimulate salt-washed vacuoles (Ungermann et al., 1999), also show some stimulation of wild-type (ACT1) vacuole fusion (Fig. 4 A, compare lane 1 with 13), but only act1157 vacuoles exhibit complete P.C. dependence (Fig. 4 A, compare lane 2 with 14). This fusion is sensitive to normal fusion inhibitors (Fig. 4 B). Immunoblot analysis was performed on purified vacuoles from ACT1, act1157, or act1101 strains (Fig. 4 C). Vacuoles from actin mutant strains have normal levels of bound proteins such as actin, calmodulin, Sec18p, Nyv1p (v-SNARE), Ypt7p (the vacuolar Rab), and Vps39p (a subunit of HOPS), and lumenal carboxypeptidase Y, showing that they are of substantially normal composition. However, the act1101 mutation causes a striking deficiency in vacuolar Cla4p, Vrp1p, Las17p, and Arp3p, though these proteins are present at normal levels in total cell extracts (Fig. 4 D). The inability of even cytosol to rescue the fusion of vacuoles from act1101 strains (Fig. 4 A, lane 18) may reflect this striking loss of actin regulatory proteins. There was also a modest diminution of vacuolar Vam3p levels (Fig. 4, C and D), which may contribute to the fusion defect of these vacuoles.
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Discussion |
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Actin is required for other yeast trafficking reactions as well. However, it is unclear in these studies whether this reflects a need for actin cytoskeleton or for actin on the surface of organelles. Actin is needed for mitochondrial motility (Simon et al., 1995) and inheritance (Hermann et al., 1997; Boldogh et al., 2001), vacuole inheritance (Hill et al., 1996), and the polarized delivery of secretory vesicles to the bud tip (Novick and Botstein, 1985; Mullholland et al., 1997; Pruyne et al., 1998). Cdc42p regulates polarized exocytosis in yeast (Pringle et al., 1995; Zhang et al., 2001) and controls actin polymerization through activation of Las17p and the Arp2/3 complex (Lechler et al., 2001). Cdc42p also interacts with proteins that mediate secretory vesicle docking and fusion in a manner that is independent of actin cytoskeleton (Adamo et al., 2001; Zhang et al., 2001). It is unclear though whether Cdc42p also controls actin-related organelle inheritance.
Actin and its regulatory proteins are required for endocytosis in yeast (Kubler and Riezman, 1993; Munn et al., 1995; Geli and Riezman, 1996) and mammals (Lamaze et al., 1997). These actin regulatory proteins include Myo5p (Geli et al., 1998), calmodulin (Kubler et al., 1994), which is also needed for vacuole homotypic fusion (Peters and Mayer, 1998), Arc35p (Schaerer-Brodbeck and Riezman, 2000b), a subunit of the Arp2/3 complex, and Pan1p, which interacts with the endocytic machinery and activates the Arp2/3 complex (Duncan et al., 2001).
Immunoblot analysis of purified vacuoles and whole cell lysate revealed an enrichment of actin regulatory proteins from Cdc42p to the Arp2/3 complex on the vacuole membrane (Fig. 2). Although the Arp2/3 complex or its subcomponents have not previously been localized to the vacuole membrane, they have been found on other subcellular organelles such as the nucleus (Weber et al., 1995; Yan et al., 1997) and the mitochondria (Boldogh et al., 2001). We now show that these proteins, which are part of a well-studied actin remodeling cascade (Higgs and Pollard, 1999), also regulate membrane fusion. Clearly the major site of Arp2/3 localization is cortical actin patches at the plasma membrane where it is involved in cell growth via regulation of vesicle delivery (Moreau et al., 1996; Lechler et al., 2001), though these studies did not preclude a continuing role in vesicle docking and membrane fusion. In addition to Arp3p and Las17p antibody inhibition, we also show that calmodulin and Las17p WCA domain, which are known to interact with and stimulate Arp2/3 function (Winter et al., 1999; Schaerer-Brodbeck and Riezman, 2000b), can stimulate membrane fusion of vacuoles which have been blocked by the addition of an NH2-terminal Las17p peptide antibody (Fig. 3 C). Additionally, specific actin mutations cause the striking loss of vacuolar localization of several actin regulatory proteins, coincident with the inability of these vacuoles to fuse in vitro (Fig. 4 C). These data thus establish a requirement for actin remodeling for vacuole fusion.
Part of the functions of calmodulin and PI(4,5)P2 in vacuole fusion may be the regulation of actin. Calmodulin mediates the signal of docking-induced calcium release (Peters and Mayer, 1998). It interacts directly with the Vo and Vtc complexes (Peters et al., 2001; Müller et al., 2002), Myo2p (Cheney et al., 1993; Brockerhoff et al., 1994), and the Arp2/3 complex (Schaerer-Brodbeck and Riezman, 2000b). Our current studies (Fig. 3 C) show that calmodulin can at least partially bypass the inhibition caused by antibody to Las17p. Genetic (Seeley et al., 2002) and biochemical (Mayer et al., 2000) data show that PI(4,5)P2 is required for vacuole fusion, perhaps in part to function as a guanine nucleotide exchange factor for Cdc42p (Zheng et al., 1996) or as an activating ligand for Las17p. PI(4)P 5-kinase has been shown to govern actin polymerization in yeast (Desrivieres et al., 1998) and mammals (Shibasaki et al., 1997).
Although our combined genetic and biochemical data clearly show that actin has a central role in vacuole fusion, the regulation of this process and the mode of actin action are unknown. Actin structure is altered by overexpression of Nrf1p (Murray and Johnson, 2000), a protein which is directly involved in vacuole fusion (Müller et al., 2002) and interacts genetically with Cdc42p (Murray and Johnson, 2000). Actin remodeling on the vacuole is regulated by the Cdc42pLas17pArp2/3 cascade. Though Cdc42p action requires the prior activation of Ypt7p (Eitzen et al., 2001), the mechanisms which connect these GTPases are unclear. Recently, we reported (Wang et al., 2002) that docked vacuoles show selective accumulation of fusion proteins at a ring around the edge of their apposed membranes and that this ring is the site of membrane fusion. Vacuolar-bound actin is also enriched at these vertices during docking (Fig. 7). Vertex enrichment is a critical subreaction of membrane fusion (Wang et al., 2002). Actin may contribute to this protein localization and might distort or destabilize the bilayer membrane on each vacuole as a prerequisite to fusion itself.
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Materials and methods |
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Protein preparation
Sec18p, Gyp7p, Gdi1p, Rdi1p, calmodulin (Cmd)1p, and IB2 were prepared as described (Brockerhoff et al., 1992; Slusarewicz et al., 1997; Eitzen et al., 2001). GST, GSTWCA, and GSTCA were expressed in Escherichia coli DH5 transformed with the vector pGEX-4T1 (Amersham Biosciences) only or with vector containing in frame BamH1EcoR1PCR products encoding the COOH-terminal 125 (WCA) or 66 (CA) amino acids of Las17p. These proteins were prepared as described for Rdi1p (Eitzen et al., 2001). Cytosol was prepared from strain K911A grown to an OD600
4, lysed by vortexing 5 min with glass beads in lysis buffer (125 mM KCl, 5 mM MgCl2, 20 mM PIPES-KOH, pH 6.8, 200 mM sorbitol, 1 mM ATP, 6.6 ng/ml leupeptin, 16.6 ng/ml pepstatin, 16.6 µM o-phenanthroline, 3.3 µM Pefabloc SC), and cleared by centrifugation (12,000 g for 10 min and then 150,000 g for 60 min at 4°C). Whole cell lysates were similarly prepared in lysis buffer from strains grown to an OD600
1 except that 1% Triton X-100 was added after glass bead vortexing and the lysate was cleared by centrifugation at 3,000 g for 4 min at 4°C. YPT7 and LAS17 peptide antibodies were raised against TEAFEDDYNDAINIRC and FEMEECFAGLLFVDINEASHC conjugated to KLH, respectively. These peptides (3 mg/ml resin) and Sec18p (10 mg/ml resin) were conjugated to Sulfolink resin (Pierce Chemical Co.) according to the manufacturer's protocol. IgG fractions were prepared from sera by protein A sepharose adsorption (Harlow and Lane, 1988). Immobilized peptides and protein were used for affinity purification and immunodepletion of antibodies. For immunodepletion experiments, IgGs (0.5 ml) were incubated with 0.1 ml packed resin in PBS for 1 h at 4°C. Flow-through samples were saved as immunodepleted fractions. The resin was washed twice with 1 ml of PBS, and bound antibodies were eluted with 0.25 ml of 100 mM glycine-HCl, pH 2.5. All samples were dialyzed into PS buffer.
Standard vacuole fusion reactions
Vacuoles were isolated from strains grown in YPD to an OD600 0.9. Fusion reactions (Haas, 1995) contain 3 µg of vacuoles lacking proteinase A and 3 µg of vacuoles lacking alkaline phosphatase in 30 µl fusion reaction buffer (FRB: 125 mM KCl, 5 mM MgCl2, 20 mM PIPES-KOH, pH 6.8, 200 mM sorbitol, 1 mM ATP, 40 mM creatine phosphate, 0.5 mg/ml creatine kinase, 10 µM CoA, 6.6 ng/ml leupeptin, 16.6 ng/ml pepstatin, 16.6 µM o-phenanthroline, 3.3 µM Pefabloc SC). Standard vacuole fusion reactions also contained P.C. (200 µg/ml calmodulin, 8 µg/ml IB2, and 1 µg/ml Sec18p). Reactions were incubated for 90 min at 27°C before assaying for alkaline phosphatase. Staging experiments with reversible blocking reagents were as described previously (Eitzen et al., 2001).
In vitro microscopic docking assay and image processing
Docking assays were performed as described previously (Wang et al., 2002) with minor modifications. Reactions (30 µl) contain 5 µg of vacuoles (from strain DKY6281) labeled with the lipophilic dye MDY-64 (3 µM; Molecular Probes). At the end of the reaction, vacuoles were mixed with 40 µl of 0.6% agarose in PS buffer. Aliquots (15 µl) were then observed by fluorescence microscopy. Images were acquired with an Olympus BX51 microscope equipped with a 60x/1.4 NA plan Apochromat objective, 100 W Mercury arc lamp, and Cooke Sensicam QE CCD camera. The microscope and camera were controlled by IP lab software (Scanalytics). An Endow GFP filter cube (Chroma Technology Corp.) was used for rhodamine-actin and Alexa Fluor 488DNaseI. The camera was operated at normal gain setting without binning. An automation script was written to facilitate reproducible image acquisition. Image processing and quantitative analysis were according to Wang et al. (2002).
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Footnotes |
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* Abbreviations used in this paper: Cmd, calmodulin; PAK, p20-activated kinase; P.C., pure components; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; WASp, Wiskott-Aldrich syndrome protein.
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Acknowledgments |
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This work was supported by grants from the National Institute of General Medical Sciences and the Human Frontier Science Program.
Submitted: 17 April 2002
Revised: 16 July 2002
Accepted: 16 July 2002
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References |
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