Functional analysis of RhoGDI inhibitory activity on vacuole membrane fusion.

RhoGDIs (Rho GDP-dissociation inhibitors) are the natural inhibitors of Rho GTPases. They interfere with Rho protein function by either blocking upstream activation or association with downstream signalling molecules. RhoGDIs can also extract membrane-bound Rho GTPases to form soluble cytosolic complexes. We have shown previously that purified yeast RhoGDI Rdi1p, can inhibit vacuole membrane fusion in vitro. In the present paper we functionally dissect Rdi1p to discover its mode of regulating membrane fusion. Overexpression of Rdi1p in vivo profoundly affected cell morphology including increased actin patches in mother cells indicative of polarity defects, delayed ALP (alkaline phosphatase) sorting and the presence of highly fragmented vacuoles indicative of membrane fusion defects. These defects were not caused by the loss of typical transport and fusion proteins, but rather were linked to the reduction of membrane localization and activation of Cdc42p and Rho1p. Subcellular fractionation showed that Rdi1p is predominantly a cytosolic monomer, free of bound Rho GTPases. Overexpression of endogenous Rdi1p, or the addition of exogenous Rdi1p, generated stable cytosolic complexes. Rdi1p structure-function analysis showed that membrane association via the C-terminal ß-sheet domain was required for the functional inhibition of membrane fusion. Furthermore, Rdi1p inhibited membrane fusion through the binding of Rho GTPases independent from its extraction activity.

Cdc42p is activated during vacuole membrane fusion in a sterol-dependent subreaction of priming.

Cdc42p is a Rho GTPase that initiates signaling cascades at spatially defined intracellular sites for many cellular functions. We have previously shown that Cdc42p is localized to the yeast vacuole where it initiates actin polymerization during membrane fusion. Here we examine the activation cycle of Cdc42p during vacuole membrane fusion. Expression of either GTP- or GDP-locked Cdc42p mutants caused several morphological defects including enlarged cells and fragmented vacuoles. Stimulation of multiple rounds of fusion enhanced vacuole fragmentation, suggesting that cycles of Cdc42p activation, involving rounds of GTP binding and hydrolysis, are required to propagate Cdc42p signaling. We developed an assay to directly examine Cdc42p activation based on affinity to a probe derived from the p21-activated kinase effector, Ste20p. Cdc42p was rapidly activated during vacuole membrane fusion, which kinetically coincided with priming subreaction. During priming, Sec18p ATPase activity dissociates SNARE complexes and releases Sec17p, however, priming inhibitors such as Sec17p and Sec18p ligands did not block Cdc42p activation. Therefore, Cdc42p activation seems to be a parallel subreaction of priming, distinct from Sec18p activity. Specific mutants in the ergosterol synthesis pathway block both Sec17p release and Cdc42p activation. Taken together, our results define a novel sterol-dependent subreaction of vacuole priming that activates cycles of Cdc42p activity to facilitate membrane fusion.

Natamycin inhibits vacuole fusion at the priming phase via a specific interaction with ergosterol.

The antifungal antibiotic natamycin belongs to the family of polyene antibiotics. Its antifungal activity arises via a specific interaction with ergosterol in the plasma membrane (te Welscher et al., J. Biol. Chem. 283:6393-6401, 2008). However, this activity does not involve disruption of the membrane barrier function, a well-known property of other members of the polyene antibiotic family, such as filipin and nystatin. Here we tested the effect of natamycin on vacuole membrane fusion, which is known to be ergosterol dependent. Natamycin blocked the fusion of isolated vacuoles without compromising the barrier function of the vacuolar membrane. Sublethal doses of natamycin perturbed the cellular vacuole morphology, causing the formation of many more small vacuolar structures in yeast cells. Using vacuoles isolated from yeast strains deficient in the ergosterol biosynthesis pathway, we showed that the inhibitory activity of natamycin was dependent on the presence of specific chemical features in the structure of ergosterol that allow the binding of natamycin. We found that natamycin inhibited the priming stage of vacuole fusion. Similar results were obtained with nystatin. These results suggest a novel mode of action of natamycin and perhaps all polyene antibiotics, which involves the impairment of membrane fusion via perturbation of ergosterol-dependent priming reactions that precede membrane fusion, and they may point to an effect of natamycin on ergosterol-dependent protein function in general.

Cdc42p and Rho1p are sequentially activated and mechanistically linked to vacuole membrane fusion.

Small monomeric GTPases act as molecular switches, regulating many biological functions via activation of membrane localized signaling cascades. Activation of their switch function is controlled by GTP binding and hydrolysis. Two Rho GTPases, Cdc42p and Rho1p, are localized to the yeast vacuole where they regulate membrane fusion. Here, we define a method to directly examine vacuole membrane Cdc42p and Rho1p activation based on their affinity to probes derived from effectors. Cdc42p and Rho1p showed unique temporal activation which aligned with distinct subreactions of in vitro vacuole fusion. Cdc42p was rapidly activated in an ATP-independent manner while Rho1p activation was kinetically slower and required ATP. Inhibitors that are known to block vacuole membrane fusion were examined for their effect on Cdc42p and Rho1p activation. Rdi1p, which inhibits the dissociation of GDP from Rho proteins, blocked both Cdc42p and Rho1p activation. Ligands of PI(4,5)P(2) specifically inhibited Rho1p activation while pre-incubation with U73122, which targets Plc1p function, increased Rho1p activation. These results define unique activation mechanisms for Cdc42p and Rho1p, which may be linked to the vacuole membrane fusion mechanism.

Stimulation of actin polymerization by vacuoles via Cdc42p-dependent signaling.

We have previously shown that actin ligands inhibit the fusion of yeast vacuoles in vitro, which suggests that actin remodeling is a subreaction of membrane fusion. Here, we demonstrate the presence of vacuole-associated actin polymerization activity, and its dependence on Cdc42p and Vrp1p. Using a sensitive in vitro pyrene-actin polymerization assay, we found that vacuole membranes stimulated polymerization, and this activity increased when vacuoles were preincubated under conditions that support membrane fusion. Vacuoles purified from a VRP1-gene deletion strain showed reduced polymerization activity, which could be recovered when reconstituted with excess Vrp1p. Cdc42p regulates this activity because overexpression of dominant-negative Cdc42p significantly reduced vacuole-associated polymerization activity, while dominant-active Cdc42p increased activity. We also used size-exclusion chromatography to directly examine changes in yeast actin induced by vacuole fusion. This assay confirmed that actin undergoes polymerization in a process requiring ATP. To further confirm the need for actin polymerization during vacuole fusion, an actin polymerization-deficient mutant strain was examined. This strain showed in vivo defects in vacuole fusion, and actin purified from this strain inhibited in vitro vacuole fusion. Affinity isolation of vacuole-associated actin and in vitro binding assays revealed a polymerization-dependent interaction between actin and the SNARE Ykt6p. Our results suggest that actin polymerization is a subreaction of vacuole membrane fusion governed by Cdc42p signal transduction.