Deletion of MSB3 and MSB4 causes a secretory defect
Because Msb3p and Msb4p display a GAP activity toward a number of Rab GTPases in vitro, a substrate promiscuity that is common among most known Rab GAPs, we decided to define the in vivo Rab target(s) of Msb3p and Msb4p by the following approaches. First, we examined possible protein-trafficking defects in cells lacking both Msb3p and Msb4p. We found that msb3
Δ cells accumulated a large number of vesicles at 24°C, whereas wild-type and single-mutant cells had none or very few vesicles ( A and A). The vesicles were 90.00 ± 11.77 nm (n
= 204) in diameter, falling within the range of 80–100 nm post-Golgi secretory vesicles (Novick and Schekman, 1979
). In most cells, the vesicles appeared to be distributed randomly in the mother and the daughter ( A, bottom left). In a few cells, vesicles were concentrated in the daughter ( A, top right). These data suggest that Msb3p and Msb4p must share a function in secretion.
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 (more ...)
Figure 4. Loss of the GAP activity of Msb3p and Msb4p causes vesicle accumulation and a defect in actin organization. (A) MSB3 msb4Δ (JGY184A), msb3-R282K msb4Δ (JGY190A), msb3Δ MSB4 (JGY51), and msb3Δ msb4-R200K (JGY127A) cells (more ...)
The second approach used to assess a possible secretory defect in msb3
Δ cells was to monitor the secretion of invertase, a sucrose-metabolizing enzyme, and of Bgl2p, an endo-β-1,3-glucanase required for cell wall organization and biogenesis (Mrsa et al., 1993
). When invertase secretion was followed over time at 24°C, the ratio of secreted invertase versus total invertase (external plus internal) was mildly, but consistently lower in msb3
Δ cells than in wild-type cells, with the difference peaking around 45 min after induction ( B). However, the majority of invertase was secreted efficiently to the periplasmic region during the course of induction (). In contrast, Bgl2p was accumulated in large quantity inside the msb3
Δ cells at both 24°C and at 37°C ( C). As expected, Bgl2p accumulation in the late secretory mutant, sec6–4
, was temperature dependent ( C). These data suggest that most vesicles accumulated in msb3
Δ cells carry Bgl2p and a small fraction of vesicles carry invertase.
Invertase secretion in wild-type and msb3Δ msb4Δ cells
Genetic evidence for the involvement of Msb3p and Msb4p in exocytosis and for Msb3p and Msb4p functioning as GAPs for Sec4p in vivo
If Msb3p and Msb4p play a role in exocytosis, deletion or overexpression of MSB3
may display genetic interactions with some of the late secretory mutants. Indeed, deletion of MSB3
produced synthetic inhibitory effects on cell growth with sec3–2
mutants at 30°C, but not with sec1–1
, and sec6–4
mutants ( A). In addition, overexpression of Msb3p or Msb4p inhibited the growth of sec2–41
cells at 30°C, but not of any other late sec
mutants, including sec1–1, sec3–2, sec4–8, sec5–24, sec6–4, sec8–9, sec9–4, sec10–2,
( B). In contrast, overexpression of Gyp1p, a GAP for Ypt1p that is involved in ER to Golgi transport but also exhibits a GAP activity toward Sec4p in vitro (Du et al., 1998
; Du and Novick, 2001
; De Antoni et al., 2002
), did not inhibit the growth of sec2–41
cells at 30°C ( B). These results suggest that Msb3p and Msb4p are involved in exocytosis and can antagonize the function of Sec2p, the known GEF for Sec4p.
Figure 2. MSB3 and MSB4 genetically interact with exocytosis genes and down-regulate SEC4 function. (A) Synthetic inhibitory effects on cell growth between msb3Δ msb4Δ and late sec mutants. Strains carrying sec3–2 (JGY32B), sec3–2 (more ...)
Because Msb3p and Msb4p display a GAP activity toward Sec4p in vitro and appear to colocalize with Sec4p at the sites of polarized growth during the cell cycle, it seemed likely that Msb3p and Msb4p might participate in the regulation of exocytosis by functioning as GAPs for Sec4p in vivo. To test this hypothesis, we took advantage of the observation that a sec4-Q79L sec15–1
double mutant is inviable at 25°C (Walworth et al., 1992
). The Q79L mutation shifts Sec4p toward its GTP-bound form by decreasing the intrinsic GTPase activity, but this Sec4p mutant is still responsive to GAP action (Walworth et al., 1992
; Du et al., 1998
). Sec15p, an effector of Sec4p, is thought to mediate the role of Sec4p in the assembly of the exocyst (Guo et al., 1999
). We reasoned that if Msb3p and Msb4p are physiological GAPs for Sec4p, their overexpression might suppress the synthetic lethality between sec4-Q79L
by decreasing the level of GTP-bound Sec4p.
To examine this possibility, we constructed a sec4-Q79L sec15–1 double mutant harboring an URA3-marked plasmid carrying wild-type SEC4. A LEU2-marked multicopy plasmid carrying either MSB3 or MSB4 was transformed into the tester strain and assayed for its ability to replace the SEC4-containing plasmid by examining cell growth on plates containing 5FOA, a chemical that selects for cells that have lost the URA3-containing plasmid ( C). Multicopy MSB3, but not GYP1 or MSB4, was able to suppress the sec4-Q79L sec15–1 mutant, supporting the hypothesis that Msb3p functions as a GAP for Sec4p in vivo.
Msb3p and Msb4p function as GAPs for Sec4p by an arginine finger–like mechanism
GAPs for Ras, Rho, and Rab GTPases all contain an invariant arginine residue (the “finger arginine”) in the catalytic domain that is critical for their GAP activities (Ahmadian et al., 1997
; Albert et al., 1999
). Because Msb3p and Msb4p contain an arginine residue (R282 in Msb3p and R200 in Msb4p) at the corresponding position, we decided to examine whether Msb3p and Msb4p function on Sec4p by a similar mechanism. In addition, we hoped that Msb3p and Msb4p mutants deficient in the GAP activity toward Sec4p might offer an opportunity to distinguish the role of Msb3p and Msb4p in secretion from their role in actin organization. For these reasons, we substituted the arginine residue in Msb3p and Msb4p for either phenylalanine or lysine and determined the properties of the mutant proteins.
To facilitate protein purification, Msb3p, Msb4p, and their derivatives were all tagged with six histidines at their COOH termini. The tagged Msb3p and the arginine mutants were expressed from a galactose-inducible promoter in yeast cells. Crude extracts containing induced Msb3p or its mutant forms were subjected to a filter assay with GTP-loaded Sec4p as a substrate. Only extracts from cells overexpressing wild-type Msb3p exhibited measurable GAP activity (unpublished data), even though the mutant proteins, Msb3p-R282F and Msb3p-R282K, and the wild-type protein were expressed at similar levels ( A). For a quantitative assay, Msb3p and its mutants were purified from induced cells by affinity chromatography and assayed for their GAP activities toward GTP-loaded Sec4p by an HPLC-based method. As shown in B, in comparison to wild-type Msb3p, both arginine mutants showed a significantly reduced Sec4p-GAP activity.
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 (more ...)
Msb4p and its mutants, Msb4p-R200F and Msb4p-R200K, were purified from E. coli cells and tested with GTP-loaded Sec4p as a substrate ( C). Again, the arginine mutation led to a significant loss of GAP activity. These data suggest that Msb3p and Msb4p function as GAPs for Sec4p by an arginine finger-like mechanism.
The GAP activity of Msb3p and Msb4p is essential for their in vivo function
Despite the drastic reduction in their GAP activity toward Sec4p, the arginine mutants of Msb3p and Msb4p were expressed at normal levels ( D) and localized to the sites of polarized growth like the wild-type proteins ( E, and unpublished data). These data suggest that a significant loss of the GAP activity of Msb3p and Msb4p does not compromise the molecular interactions required for their targeting to the growth sites.
To determine whether the GAP activity of Msb3p and Msb4p is required for their in vivo function, we developed two assays. The first assay is based on our previous observation that msb3
Δ quadruple mutant is inviable with a loss-of-polarity phenotype (Bi et al., 2000
). The second assay is based on our new observation that msb3
Δ is synthetically lethal with cdc42–201
, a newly isolated temperature-sensitive cdc42
allele (Zhang et al., 2001
). These two tester strains were kept alive by introducing an URA3
-marked plasmid that carries either wild-type GIC1
for the first assay or CDC42
for the second assay. HA-tagged Msb3p, Msb4p, and their arginine mutants expressed from a LEU2
-marked, high-copy plasmid in the tester strains were assayed for their ability to replace the URA3
-marked plasmids on SC-Leu+5FOA plates. Plasmids carrying the arginine mutants of MSB3
failed to replace the URA3
-marked plasmids in both assays, in direct contrast to the plasmids carrying wild-type MSB3
( F and unpublished data). These data indicate that the GAP activity of Msb3p and Msb4p is essential for their in vivo function(s).
Overexpression of Msb3p-R282K and Msb4p-R200K mutants also failed to inhibit the growth of sec2–41 cells at 32°C ( G), suggesting that the GAP activity of Msb3p and Msb4p is required for antagonizing the function of Sec2p. The arginine mutant of Msb3p also failed to suppress the synthetic lethality between sec4-Q79L and sec15–1 ( H), further supporting the notion that it is the GAP activity of Msb3p toward Sec4p, not merely the presence of Msb3p, that is responsible for the suppression.
The GAP activity of Msb3p and Msb4p is required for efficient exocytosis and polarized actin organization
To determine whether vesicle accumulation in msb3Δ msb4Δ cells was due to the absence of the proteins or the loss of their GAP activity, we examined two pairs of haploid strains, JGY184A (MSB3 msb4Δ) and JGY190A (msb3-R282K msb4Δ), and JGY51 (msb3Δ MSB4) and JGY127A (msb3Δ msb4-R200K). In most MSB3 msb4Δ or msb3Δ MSB4 cells, either no or just a few vesicles (usually <10 vesicles per cell section) were detected ( A, left). In contrast, most msb3-R282K msb4Δ or msb3Δ msb4-R200K cells accumulated a large number of vesicles similar to those observed in msb3Δ msb4Δ cells ( A, right). These data indicate that the loss of the GAP activity of Msb3p and Msb4p is responsible for vesicle accumulation.
Cells of msb3
Δ strain are rounder in shape, heterogeneous in size, and have a partially disrupted actin cytoskeleton (Bi et al., 2000
). Actin patches in these cells tended to delocalize into the mother side at early stages of the cell cycle when the patches should be predominantly concentrated in the buds ( B, compare columns 1 and 2). Actin cables were clearly present and largely well organized in msb3
Δ cells. However, some cables appeared to be shorter, and sometimes misoriented in the mutant strain ( B, compare columns 1 and 2).
To determine whether the GAP activity of Msb3p and Msb4p is required for actin-patch organization, MSB3, msb3-R282K, MSB4, or msb4-R200K was integrated into the msb3Δ msb4Δ mutant at the msb3Δ or msb4Δ locus, respectively. The integrants with MSB3 or MSB4 showed normal cell morphology and actin-patch organization ( B, columns 3 and 5). Interestingly, the integrants with msb3-R282K or msb4-R200K displayed similar defects in cell morphology and actin-patch organization as the msb3Δ msb4Δ mutant did ( B, columns 4 and 6), suggesting that the GAP activity of Msb3p and Msb4p is required for actin-patch organization.
is known to suppress the growth defect of cdc42–1
cells at the nonpermissive temperature (Bi et al., 2000
) ( A, top). We found that multicopy MSB3
also suppressed the growth defect of another cdc42-
Ts allele, cdc42–201
( A, bottom). In addition, the budding and the actin-organization defects in both cdc42-
Ts mutants were largely suppressed by multicopy MSB3
( B) (). Interestingly, multicopy msb3-R282K
failed to suppress both the budding and the actin-organization defects of the two cdc42-
Ts mutants () (), which were not defective in secretion per se as indicated by EM studies ( C). These results suggest that the GAP activity of Msb3p is required for the suppression of the actin-organization defects in cdc42
mutants. Together with the results described in the previous section, these data raise an intriguing possibility that polarized secretion may be normally involved in modulating polarized actin organization.
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 (more ...)
The GAP activity of Msb3p is required for the suppression of the actin-organization defects in cdc42-Ts mutants
A defect in actin-patch organization can be a consequence of a primary defect in polarized secretion
The fact that the GAP activity of Msb3p is required to rescue both the secretory and the actin-patch-organization defects in msb3
Δ cells raises the possibility that the actin-patch disorganization in msb3
Δ cells might be a consequence of the fusion of mistargeted vesicles with the plasma membrane of the mother cells. To examine this possibility, we took advantage of mutants in TPM1
, which encode two isoforms of tropomyosin that are required specifically for the formation of actin cables but not actin patches (Pruyne et al., 1998
). When tropomyosins are conditionally inactivated, all actin cables are lost within one minute. As a result, secretory vesicles are no longer transported to the daughter cell, but instead fuse with the plasma membrane of the mother cell. After inactivation of tropomyosins for 30–60 min, actin patches become randomly distributed in both the mother and the daughter cells () (Pruyne et al., 1998
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 tpm2Δ (ABY973), tpm1–2 tpm2Δ (ABY971), sec6–4 (more ...)
To test our hypothesis, we examined the distribution of actin patches in sec6–4 tpm1–2 tpm2Δ cells. At the restrictive temperature, secretory vesicles in this mutant are no longer delivered to the bud due to the loss of actin cables. In addition, vesicles in this mutant fail to fuse with the plasma membrane due to the inactivation of Sec6p, a component of the exocyst that is essential for vesicle tethering. We observed that, upon shifting to 36°C for 60 min, 51% of the triple mutant cells still displayed a polarized organization of actin patches in comparison to 88% of the tpm2Δ cells, 85% of the sec6–4 cells, and 0% of the tpm1–2 tpm2Δ cells (). These results suggest that the actin-patch disorganization in tpm1–2 tpm2Δ cells, and, by extrapolation, in msb3Δ msb4Δ cells, depends on the fusion of secretory vesicles in the mother cells with the plasma membrane.
One possible explanation for the polarized organization of actin patches in sec6–4 tpm1–2 tpm2Δ cells is that the lifespan of the “old patches” (existed before the temperature shift) in the buds of the small-budded cells is significantly increased. We measured the lifespan of actin patches in four different strains at two different temperatures, 20°C and 36°C, using Abp1p-GFP as a marker for the patches (). At 20°C, actin patches in all four strains displayed a similar lifespan, ~16 s. At 36°C, the lifespan of actin patches in both tropomyosin mutants (tpm2Δ and tpm1–2 tpm2Δ) were ~9 s. In contrast, the lifespan of actin patches in both mutants carrying sec6–4 (sec6 tpm1–2 tpm2Δ, and sec6–4) were ~21 s. We also observed that the lifespan of actin patches in the mother and the daughter compartments of the same cell were virtually identical for all four strains at both temperatures. These data suggest that blocking exocytosis at 36°C increases the lifespan of actin patches, but this increase alone is not sufficient to explain the polarized actin-patch organization in the sec6 tpm1–2 tpm2Δ mutant.