Time-lapse analysis of vacuole inheritance. Time-lapse analysis. PTC1 is required for movement of the vacuole into the bud. Time-lapse images of wild-type (A) and ptc1Δ (B) cells grown at room temperature were acquired at 2-min intervals. Scale bar, 1 μm.

PTC1 is required for the proper distribution of secretory vesicles and peroxisomes. (A) PTC1 is required for secretory vesicle movement. DIC (a and f), FM4-64 (vacuole; b and g), GFP-Sec4p (secretory vesicle; c and h), and merged images (d, e, i, and j) of wild-type (a–e) and ptc1Δ (f–j) cells. (B)The ptc1Δ mutant is temperature sensitive for growth. Cells grown at the indicated temperatures for 2 d on YEPD plates. (C) PTC1 is involved in peroxisome distribution. DIC image (a, f, and k), FM4-64 (vacuole; b, g, and l), GFP-PTS1 (peroxisome; c, h, and m), and merged images (d, e, i, j, n, and o) of wild-type (a–e), myo2-66 (f–j), and ptc1Δ (k–o) cells. Right panel, quantitative analysis of peroxisome distribution in wild-type, myo2-66, and ptc1Δ cells. Peroxisome inheritance was assessed in cells with buds that were less than half the diameter of the mother. Error bars, SDs calculated from five experiments.

PTC1 is required for the proper localization of both myosin-V motors. (A) Myo4p-Venus is mis-localized in ptc1Δ cells. DIC image (a and e), FM4-64 (vacuole; b and f), Myo4p-Venus (c and g), and merged images (d and h) of wild-type (a–d) and ptc1Δ (e–h) cells. Graph shows quantitative analysis of Myo4p-Venus localization in wild-type and ptc1Δ cells. Small- (∼90), medium- (∼220), and large- (∼130) budded cells were classified. (B) Myo2p-Venus is partially mis-localized in ptc1Δ cells. DIC image (a and e), FM4-64 (vacuole; b and f), Myo2p-Venus (c and g), and merged images (d and h) of wild-type (a–d) and ptc1Δ (e–h) cells. Graph shows quantitative analysis of Myo2p-Venus localization in wild-type and ptc1Δ cells. Small- (∼90), medium- (∼200), and large- (∼110) budded cells were classified. The schematics below the graphs illustrate the possible locations of the Venus-tagged myosin-V motors: the bud tip; diffusely spread through the bud, or the mother-bud neck. Blue asterisks indicate a decreased percentage of ptc1Δ compared with wild-type cells, and red asterisks indicate increased percentage of ptc1Δ compared with wild-type cells. (C) Total cell lysates of MYO4-Venus (lane 1), MYO4-Venus/ptc1Δ (lane 2), and wild type (lane 3) were analyzed by immunoblot with antibodies directed against GFP or Pgk1p (loading control). (D) Total cell lysates of MYO2-Venus (lane 1), MYO2-Venus/ptc1Δ (lane 2), and wild type (lane 3) were analyzed by immunoblot with antibodies directed against GFP or Pgk1p (loading control).

PTC1 regulates the association of Myo2p and Vac17p. (A) In wild-type cells, Vac17p-3xGFP is localized to the vacuole membrane and is concentrated at the leading edge of the vacuole. In ptc1Δ, Vac17p-3xGFP was localized on the vacuole membrane, but was not concentrated at the leading edge of the vacuole. DIC image (a and h), FM4-64 (vacuole; b, e, i, and l), Vac17p-3xGFP (c, f, j, and m), and merged images (d, g, k, and n) of wild-type (a–g) and ptc1Δ (h–n) cells. (B) A fusion protein of full-length Myo2p and Vac17p suppressed the defect in vacuole inheritance in a ptc1Δ strain. Quantitative analysis of vacuole inheritance in ptc1Δ cells with pRS415 (vector control), pRS415 VAC17, pRS415 MYO2, pRS415 myo2-N1304S, pRS415 MYO2-VAC17, or pRS415 myo2-N1304S-VAC17. Error bars, SDs calculated from at least three experiments. (C) Total cell lysates of ptc1Δ with pRS415 (vector control, lane 1), pRS415 MYO2-VAC17 (lane 2), pRS415 myo2-N1304S-VAC17 (lane 3), pRS415 VAC17 (lane 4), pRS415 MYO2 (lane 5), and pRS415 myo2-N1304S (lane 6) were analyzed by immunoblot with antibodies directed against Vac17p or Pgk1p (loading control). Note that the levels of the Myo2p-Vac17p fusion protein are lower than Vac17p levels. Thus the suppression results from the fusion protein, not from an increase in Vac17p.

PTC1 functions in vacuole inheritance. (A) vac10-1 and ptc1Δ cells have a vacuole inheritance defect and fragmented vacuoles. Wild-type (a–c), vac10-1 (d–f), and ptc1Δ (g–i) cells were labeled with the vacuole-specific dye FM4-64. Scale bar, 2 μm. (B) Quantitative analysis of vacuole inheritance in wild-type, vac10-1, and ptc1Δ cells. Vacuole inheritance assessed as the percent cells with an inherited vacuole in the bud. □, normal vacuole inheritance; ■, bud without detectable FM4-64; ▩, weaker FM4-64 signal in the bud than in the mother cell. (C) The vac10-1 mutant is due to a missense mutation in PTC1. The ptc1-D272N mutant does not complement vac10-1. (B and C) Error bars, SDs calculated from three experiments.

The phosphatase activity of Ptc1p is required for vacuole inheritance. (A) Quantitative analysis of vacuole inheritance in ptc1Δ cells with pRS416 PTC1, vector control (pRS416), pRS416 GFP-PTC1, pRS416 GFP-ptc1-D58N, pRS416 GFP-ptc1-D272N, pRS416 GFP-ptc1-E35A/D36A, or pRS416 GFP-ptc1-D233A. More than 100 cells were counted. (B) Total cell lysates of ptc1Δ with pRS416 PTC1 (lane 1), vector control (lane 2), pRS416 GFP-PTC1 (lane 3), pRS416 GFP-ptc1-D58N (lane 4), pRS416 GFP-ptc1-D272N (lane 5), pRS416 GFP-ptc1-E35A/D36A (lane 6), and pRS416 GFP-ptc1-D233A (lane 7) were analyzed by immunoblot with antibodies directed against GFP (for GFP-Ptc1p), Vac17p, or Pgk1p (loading control).

PTC1 is required for the proper distribution of ASH1 mRNA, but not late-Golgi inheritance and mitotic spindle orientation. (A) PTC1 is not required for distribution of the late-Golgi. DIC image (a, f, and k), FM4-64 (vacuole; b, g, and l), Sec7p-3xGFP (late-Golgi; c, h, and m), and merged images (d, e, i, j, n, and o) of wild-type (a–e), myo2-66 (f–j), and ptc1Δ (k–o) cells. Right panel, quantitative analysis of late-Golgi distribution in wild-type, myo2-66, and ptc1Δ cells. Late-Golgi inheritance was assessed in cells with buds less than one-third the diameter of the mother. (B) PTC1 is not required for mitotic spindle orientation. DIC image (a, d, and g), GFP-Tub1p (b, e, and h), and merged images (c, f, and i) of wild-type (a–c), kar9Δ (d–f), and ptc1Δ (g–i) cells. Right panel, quantitative analysis of mitotic spindle orientation in wild-type, kar9Δ, and ptc1Δ cells. (C) PTC1 is involved in ASH1 mRNA localization. MS2-GFP and RNA with the ASH1 3′ UTR and MS2-binding sites were coexpressed in wild-type (a–c) or ptc1Δ (d–f) cells. DIC (a and d), MS2-GFP (ASH1 reporter; b and e), and merged images (c and f). Right panel, quantitative analysis of ASH1 mRNA localization in wild-type and ptc1Δ cells. Error bars, SDs calculated from three (A) or four (B and C) experiments.

Ptc1p is required to maintain the steady-state levels of Vac17p. (A) Steady-state levels of Mmr1p and Inp2p were reduced in cells lacking PTC1. Total cell lysates of wild-type (lanes 1 and 3) and ptc1Δ cells (lanes 2 and 4) were analyzed by immunoblot with antibodies directed against GFP or Pgk1p (loading control). (B) Steady-state levels of Vac17p are reduced in cells lacking PTC1. Total cell lysates of vac17Δ (lane 1), wild type (lane 2), and ptc1Δ (lane 3) were analyzed by immunoblot with antibodies directed against the Myo2p-tail, Vac8p, Vac17p, or Pgk1p (loading control). (C) Semiquantitative RT-PCR. Levels of VAC17 mRNA in ptc1Δ are the same as the levels in wild-type cells. PGK1 mRNA was used as loading control. Lanes 1 and 2, no reverse transcriptase (−RT); lanes 3–8, +RT. (D) Steady-state levels of Vac17p are also reduced in cells lacking NBP2. Total cell lysates of wild-type (lane 1), ptc1Δ (lane 2), and nbp2Δ cells (lane 3) were analyzed by immunoblot with antibodies directed against Vac17p or Pgk1p (loading control). (E) Overexpression of VAC17 in ptc1Δ cells partially restored vacuole inheritance but not vacuole fragmentation. A 2μ vector (pRS426; a–c) or 2μ-VAC17 (d–f) expressed in ptc1Δ cells, labeled with FM4-64. (F) Quantitative analysis of vacuole inheritance in ptc1Δ cells with 2μ (vector control) or 2μ-VAC17. Error bars, SDs from three experiments. (G) Vac17p was overexpressed from a 2μ vector. Total cell lysates of ptc1Δ with CEN plasmid (pRS416; vector control, lane 1), 2μ plasmid (pRS426; vector control, lane 2), CEN-VAC17 (lane 3), 2μ-VAC17 (lane 4), CEN-PTC1 (lane 5), and 2μ-PTC1 (lane 6) were analyzed by immunoblot with antibodies directed against Vac17p or Pgk1p (loading control).

Model of PTC1-NBP2 regulation of multiple myosin-V cargoes. We propose that PTC1-NBP2 regulates the formation of Myo2p complexes through as yet undetermined pathway(s). PTC1-NBP2 regulates Myo4p function through the CWI pathway (Du et al., 2006). Myo2p forms a dimer (Beningo et al., 2000), and Myo4p forms a single-head motor (Dunn et al., 2007; Heuck et al., 2007; Hodges et al., 2008). The location of cargo attachment to Myo4p is currently unknown. H, head domain; T, tail domain; R, receptor protein(s), C, cargo.