The GAP activity of Msb3p and Msb4p for the Rab GTPase Sec4p is required for efficient exocytosis and actin organization - PubMed (original) (raw)

The GAP activity of Msb3p and Msb4p for the Rab GTPase Sec4p is required for efficient exocytosis and actin organization

Xiang-Dong Gao et al. J Cell Biol. 2003.

Abstract

Polarized growth in Saccharomyces cerevisiae is thought to occur by the transport of post-Golgi vesicles along actin cables to the daughter cell, and the subsequent fusion of the vesicles with the plasma membrane. Previously, we have shown that Msb3p and Msb4p genetically interact with Cdc42p and display a GTPase-activating protein (GAP) activity toward a number of Rab GTPases in vitro. We show here that Msb3p and Msb4p regulate exocytosis by functioning as GAPs for Sec4p in vivo. Cells lacking the GAP activity of Msb3p and Msb4p displayed secretory defects, including the accumulation of vesicles of 80-100 nm in diameter. Interestingly, the GAP activity of Msb3p and Msb4p was also required for efficient polarization of the actin patches and for the suppression of the actin-organization defects in cdc42 mutants. Using a strain defective in polarized secretion and actin-patch organization, we showed that a change in actin-patch organization could be a consequence of the fusion of mistargeted vesicles with the plasma membrane.

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Figures

Figure 1.

Figure 1.

Deletion of MSB3 and MSB4 causes a secretory defect. (A) Wild-type (YEF473) and msb3Δ msb4Δ (YEF1631) cells were grown in YPD media at 24°C and processed for electron microscopy. Bars, 0.5 μm. (B) Invertase secretion. Wild-type (YEF473A) and msb3Δ msb4Δ (YEF1289) cells were induced for secretion of invertase at 24°C. The percentage of external (Ext, secreted) pool versus total invertase (Ext + Int) was measured at indicated times after induction. (C) Bgl2p secretion. Wild-type (YEF473A), msb3Δ msb4Δ (YEF1289) and sec6–4 (BY37) cells were grown at 24°C, or shifted to 37°C for 1 h. The amounts of internal and external pools of Bgl2p were analyzed by Western blotting with anti-Bgl2p antibody. Samples of external pool were loaded only half the amount as those of internal pool.

Figure 2.

Figure 2.

MSB3 and MSB4 genetically interact with exocytosis genes and down-regulate SEC4 function. (A) Synthetic inhibitory effects on cell growth between _msb3_Δ _msb4_Δ and late sec mutants. Strains carrying sec3–2 (JGY32B), _sec3–2 msb3_Δ _msb4_Δ (JGY37B), sec9–4 (JGY31B), _sec9–4 msb3_Δ _msb4_Δ (JGY40A), sec6–4 (JGY30A), and _sec6–4 msb3_Δ _msb4_Δ (JGY39A) were streaked onto YPD plates and incubated at 24°C for 3 d, or at 30°C for 2 d. (B) Multicopy MSB3 or MSB4 inhibits the growth of sec2–41 cells. The sec2–41 strain (JGY28B) and the control sec6–4 strain (JGY30A) carrying plasmids YEplac181 (Vector), YEp181-MSB3, YEp181-MSB4, and YEp181-GYP1 were streaked onto SC-Leu plates and incubated at 24°C for 3 d, or at 30°C for 2 d. (C) Multicopy MSB3 suppresses the lethality of the sec15–1 s4-Q79L double mutant. YEplac181 alone, or carrying 3HA-tagged MSB3 or MSB4, or GYP1, was transformed into strain JGY86A (sec15–1 s4-Q79L, pRS316-SEC4). Transformants were replica-plated onto a SC-Leu+5FOA plate and incubated at 24°C for 3 d.

Figure 3.

Figure 3.

Msb3p and Msb4p function as GAPs for Sec4p by an arginine finger-like mechanism. (A) Crude extracts from yeast cells overexpressing His6-tagged Msb3p wild-type (WT) or arginine mutants were analyzed by Western blotting with anti-His6 antibody. (B and C) Time course of hydrolysis of Sec4p-bound GTP stimulated by the wild-type or the arginine mutants of Msb3p (B) or by the wild-type or the arginine mutants of Msb4p (C). (D) The arginine mutants of Msb3p and Msb4p express normally. Yeast strain JGY18 (_msb3_Δ _msb4_Δ cdc42–201, pRS316-CDC42) carrying plasmid YEp181–3HA-MSB3, YEp181–3HA-MSB3-R282F, YEp181–3HA-MSB3-R282K, YEp181–3HA-MSB4, YEp181–3HA-MSB4-R200F, or YEp181–3HA-MSB4-R200K was analyzed for the expression of wild-type and the arginine mutants of Msb3p and Msb4p. The mitochondrial outer membrane protein Isp42p was used as a loading control. (E) The arginine mutants of Msb3p localize normally. YEp181–3HA-MSB3, YEp181–3HA-MSB3-R282F, or YEp181–3HA-MSB3-R282K was transformed into YEF1619 (_msb3_Δ/_msb3_Δ). Transformants were grown at 24°C and processed for immunofluorescence with anti-HA antibody. (F) The GAP activity of Msb3p and Msb4p is required for their in vivo function. YEplac181 alone, or carrying 3HA-tagged MSB3, MSB4, or their respective arginine mutants, was transformed into strain JGY18 (_msb3_Δ _msb4_Δ cdc42–201, pRS316-CDC42). Transformants were replica plated onto a SC-Leu+5FOA plate and incubated at 24°C for 4 d. (G) The GAP activity of Msb3p and Msb4p is required for their inhibitory effects on a sec2–41 mutant. YEplac181 alone, or carrying 3HA-MSB3, 3HA-msb3-R282K, 3HA-MSB4, 3HA-msb4-R200K was transformed into JGY28B (sec2–41). Transformants were streaked onto SC-Leu plates and incubated for 3 d at 24°C, or for 2 d at 32°C. (H) The GAP activity of Msb3p is required for the suppression of the sec15–1 s4-Q79L mutant. YEplac181 alone, or carrying 3HA-MSB3 or 3HA-msb3-R282K, was transformed into strain JGY86A (sec15–1 s4-Q79L, pRS316-SEC4). Transformants were replica plated onto a SC-Leu+5FOA plate and incubated at 24°C for 3 d.

Figure 4.

Figure 4.

Loss of the GAP activity of Msb3p and Msb4p causes vesicle accumulation and a defect in actin organization. (A) _MSB3 msb4_Δ (JGY184A), _msb3-R282K msb4_Δ (JGY190A), _msb3_Δ MSB4 (JGY51), and _msb3_Δ msb4-R200K (JGY127A) cells were grown in YPD media at 24°C and processed for electron microscopy. Please note that the appearance of vesicles varies from batch to batch due to possible variations in sample preparations. Bars, 0.5 μm. (B) Wild-type (YEF473), _msb3_Δ _msb4_Δ (YEF1631), _MSB3 msb4_Δ (JGY184), _msb3-R282K msb4_Δ (JGY190), _msb3_Δ MSB4 (JGY71), and _msb3_Δ msb4-R200K (JGY130) diploid cells were grown at 24°C and stained for F-actin.

Figure 5.

Figure 5.

The GAP activity of Msb3p is required for the suppression of the budding and the actin-organization defects in _cdc42-_Ts mutants. (A) YEplac181 alone, or carrying 3HA-MSB3 or 3HA-msb3-R282K or 3HA-MSB1 (MSB1, a known multicopy suppressor of cdc42–1 [Bender and Pringle, 1989], serves as a control here), was individually transformed into strains YEF115 (cdc42–1) and YEF2258 (cdc42–201), respectively. Transformants were streaked onto SC-Leu plates and incubated for 3 d at 24°C, or for 2 d at 38°C (for cdc42–1 host) and at 35.5°C (for cdc42–201 host). (B) The unbudded population of cdc42–201 cells carrying YEplac181, YEp181–3HA-MSB3, or YEp181–3HA-MSB3-R282K were enriched and released into SC-Leu medium at 35.5°C. Cells were fixed and stained for F-actin and DNA. Representative cells before shifting (24°C) and after shifting (35.5°C 6 h) were shown (see also Table II). (C) YEF115 (cdc42–1) and YEF2258 (cdc42–201) cells were grown in YPD media at 24°C, and then shifted to 37.5°C and 36°C for 1 h, respectively. Cells were processed for electron microscopy. Bars, 0.5 μm.

Figure 6.

Figure 6.

Blocking of vesicle fusion with the plasma membrane prevents the reorganization of actin patches into the mother compartment in cells lacking actin cables. (A and B) Strains _tpm2_Δ (ABY973), _tpm1–2 tpm2_Δ (ABY971), _sec6–4 tpm1–2 tpm2_Δ (ABY999), and sec6–4 (JGY381) carrying GFP-TUB1 were grown in YPD media at 24°C and then shifted to 36°C for 0, 30, and 60 min. Cells were fixed and stained for F-actin. (A) The percentage of small-budded cells that displayed a polarized distribution of actin patches after shifting to 36°C for different periods of time was shown. (B) Representative cells shifted to 36°C for 60 min were shown. (C) The same strains as in A, except carrying ABP1-GFP instead of GFP-TUB1, were grown at 20°C. The lifespan (in seconds) of actin patches in each strain was measured in time-lapse series at 20°C, and at 36°C after shifting to 36°C for 60 min. At least 100 actin patches from both the mother and the daughter compartments of budded cells were analyzed for each strain at each temperature. (D) The percentage distribution of actin-patch lifespan in strains _tpm1–2 tpm2_Δ (ABY971) and _sec6–4 tpm1–2 tpm2_Δ (ABY999) at 36°C.

Figure 7.

Figure 7.

Models on the spatial regulation of exocytosis and on the coupling of exocytosis to actin-patch organization. (A) Role of Msb3p and Msb4p in spatial regulation of exocytosis. Secretory vesicles carrying Sec4p-GTP, Sec2p (GEF for Sec4p), and actin-patch clustering and/or assembly factor X are delivered to the bud tip by Myo2p (a type V myosin) along actin cables. Msb3p and Msb4p at the bud tip stimulate the hydrolysis of Sec4p-bound GTP, leading to the recycling of Sec4p for next round of exocytosis. The cargo molecule X, which could include the polarity proteins Cdc42p, Rho1p, and the actin-binding protein Aip3p/Bud6p, is delivered to the bud tip to reenforce the polarized organization of actin patches, which, in turn, may be involved in the recycling of the exocytic machinery such as the exocyst for next round of secretion. (B) Fusion of an exocytic vesicle with the plasma membrane leads to the formation and/or trapping of an actin patch at the fusion site. In _msb3_Δ _msb4_Δ cells, vesicles that are not transported out of the mother compartment can fuse with the plasma membrane (not depicted in the diagram). Vesicles that are not tethered at the bud tip (green arrows) can diffuse into the mother side and fuse with the plasma membrane, depositing their cargoes, including factor X, at the fusion sites for the generation and/or trapping of actin patches. In a tropomyosin mutant (black arrow), the vesicles are not transported to the bud and thus fuse with the plasma membrane of the mother compartment, leading to the reorganization of actin patches as described for _msb3_Δ _msb4_Δ cell.

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