Remodeling of organelle-bound actin is required for yeast vacuole fusion

Abstract

Actin participates in several intracellular trafficking pathways. We now find that actin, bound to the surface of purified yeast vacuoles in the absence of cytosol or cytoskeleton, regulates the last compartment mixing stage of homotypic vacuole fusion. The Cdc42p GTPase is known to be required for vacuole fusion. We now show that proteins of the Cdc42p-regulated actin remodeling cascade (Cdc42p --> Cla4p --> Las17p/Vrp1p --> Arp2/3 complex --> actin) are enriched on isolated vacuoles. Vacuole fusion is dramatically altered by perturbation of the vacuole-bound actin, either by mutation of the ACT1 gene, addition of specific actin ligands such as latrunculin B or jasplakinolide, antibody to the actin regulatory proteins Las17p (yeast Wiskott-Aldrich syndrome protein) or Arp2/3, or deletion of actin regulatory genes. On docked vacuoles, actin is enriched at the "vertex ring" membrane microdomain where fusion occurs and is required for the terminal steps leading to membrane fusion. This role for actin may extend to other trafficking systems.

Figure 1.

A signaling pathway which regulates actin remodeling. Arrows depict known protein interactions. Dashed lines and arrows depict pathways seen in mammalian cells. Lines show other interacting factors.

Figure 2.

Actin affects vacuole morphology. (A) Vacuole morphology of gene deletion or actin or LAS17 mutant strains. The strains RLY 574 (arp2Δ), DDY2266 (las17–16), GEY1290 (myo3Δ), GEY1090 (myo5Δ), GEY4840 (sac2Δ), GEY1290 (sac6Δ), GEY3370 (vrp1Δ), GEY2980 (cla4Δ), GEY3700 (arc18Δ), GEY0394–101 (act1–101), GEY0394–157 (act1–157), and the wild-type strain BY4742 (WT) were grown overnight in YPD, stained with FM4–46 and examined for vacuole morphology. The width of the field shown in each of the nine panels is 25 μm. (B) Immunoblot analysis of vacuoles and whole cell lysate prepared from strain GEY6024 for actin, actin signaling proteins (Vrp1p, Las17p, Arp3p, Cla4p, and Cdc42p), vacuolar proteins (Ypt7p and CPY), and a cytosolic chaperone (Ssa1p).

Figure 3.

Actin regulatory proteins are required for vacuole fusion. (A) Las17p antibodies inhibit vacuole fusion. Standard fusion reactions were incubated at 27°C in the presence of increasing concentrations of anti-LAS17 peptide antibodies (left) or anti-YPT7 peptide antibodies (right) that were either untreated (▴) or were immunodepleted by incubation with immobilized LAS17 peptide (×) or immobilized YPT7 peptide (□) (as described in Materials and methods). Titration curves for immunodepleted samples were normalized by volume to the nonimmunodepleted samples. (B) Las17p antibody no longer inhibits after docking. Standard fusion reactions (7×) were first incubated with either 50 μg/ml anti-Sec18p, 64 μg/ml anti-Ypt7 antibodies, or 3.5 mM BAPTA for 30, 35, or 45 min, respectively. Sec18p (10 μg/ml), Ypt7p peptide (30 μg/ml), or 4 mM calcium was then added to reverse these blocks, and aliquots (30 μl) were immediately distributed into tubes containing the indicated second inhibitors and incubated at 27°C (lanes 2–7) or on ice (lane 1) for 90 min total. (C) Bypass of Las17p function. Fusion reactions with 8 μg/ml IB2 and 1 μg/ml Sec18p were incubated with increasing concentrations of anti-Las17 antibodies in the presence or absence of 1 mg/ml calmodulin, 0.5 mg/ml GST, 0.5 mg/ml GST–WCA, or 0.5 mg/ml GST–CA. (D) Arp3p antibodies inhibit vacuole fusion. Standard fusion reactions were incubated with an increasing amount of NH₂- and COOH-terminal Arp3p antibodies which had been dialyzed into PS buffer and used directly or heat denatured by incubation at 95°C for 5 min.

Figure 4.

Actin mutations impair vacuole fusion. (A) Vacuoles isolated from strains bearing wild-type actin (GEY0394/0398), act1–157 (GEY0394–157/0398–157), or act1–101 (GEY0394–101/0398–101) were tested for fusion in the absence of additional soluble components (None) or in the presence of IB2, calmodulin, Sec18p, P.C. (IB2, Sec18p, and Cmd1p), or cytosol (0.5 mg/ml). (B) Standard fusion reactions of ACT1 or act1–157 vacuoles are blocked by inhibitors of the normal fusion pathway. (C) Immunoblot analysis of purified vacuoles (5 μg/lane) isolated from strain GEY0394 (ACT1) or the actin mutant strains GEY0394–157 (act1–157), and GEY0394–101 (act1–101). (D) Immunoblot analysis of whole cell lysates (30 μg/lane) from the strains in C.

Figure 5.

Actin ligands inhibit vacuole fusion. (A) Inhibition of vacuole fusion by latrunculin B and jasplakinolide. Drugs were added to standard fusion reactions. (B) Standard vacuole fusion reactions were arrested while undocked by incubation with 64 μg/ml Ypt7 antibodies or when docked (but not fused) by incubation with 3.5 mM BAPTA for 35 or 45 min, respectively. The indicated second inhibitors were added after reversal of the initial blocks by the addition of either 30 μg/ml YPT7 peptide or 4 mM calcium, respectively. (C) Kinetic analysis of inhibition by latrunculin B. A large standard fusion reaction was incubated at 27°C. At various times, 30-μl aliquots were placed on ice (defining the fusion curve) or added to tubes containing anti-Sec18p antibodies (priming curve), anti-Ypt7p antibodies (docking curve), or latrunculin B (300 μM final concentration), and further incubated at 27°C for 90 min total.

Figure 6.

Actin remodeling is not needed for Ypt7-dependent docking. The quantitative microscopic assay of vacuole docking was performed as described (Wang et al., 2002). Approximately 700 vacuoles were counted for each reaction, and the percentage of vacuoles in each cluster size was scored. Gdi1p (100 μg/ml), jasplakinolide (250 μM), and latruculin B (400 μM) were added as indicated.

Figure 7.

Actin is enriched at the vertices of docked vacuoles. Purified vacuoles were either docked by the physiological Ypt7p-dependent pathway or clustered by cosedimentation as described in Wang et al. (2002). (A) Rhodamine-actin (4 μM, total rabbit muscle actin) was added at the beginning of the reaction, or Alexa Fluor 488–DNaseI (0.2 μM) was added to docked or cosedimented vacuoles (B). After a 30-min incubation at 27°C, vacuoles were fixed with 1% formaldehyde for 20 min at room temperature, sedimented at 3000 g for 2 min, resuspended in PS buffer, and subjected to microscopic analysis (as described in Materials and methods). Ratiometric images of representative clusters are shown. (C) Cumulative distribution plots of the ratios of pixel values (rhodamine:MDY-64 lipid label) obtained for each morphometric domain (vertex, boundary, and outside edge midpoints) and each treatment. Each curve represents an average of 410 individual ratio measurements on 110–115 vacuole clusters from three independent experiments.