The Rho GDI Rdi1 Regulates Rho GTPases by Distinct Mechanisms

Abstract

The small guanosine triphosphate (GTP)-binding proteins of the Rho family are implicated in various cell functions, including establishment and maintenance of cell polarity. Activity of Rho guanosine triphosphatases (GTPases) is not only regulated by guanine nucleotide exchange factors and GTPase-activating proteins but also by guanine nucleotide dissociation inhibitors (GDIs). These proteins have the ability to extract Rho proteins from membranes and keep them in an inactive cytosolic complex. Here, we show that Rdi1, the sole Rho GDI of the yeast Saccharomyces cerevisiae, contributes to pseudohyphal growth and mitotic exit. Rdi1 interacts only with Cdc42, Rho1, and Rho4, and it regulates these Rho GTPases by distinct mechanisms. Binding between Rdi1 and Cdc42 as well as Rho1 is modulated by the Cdc42 effector and p21-activated kinase Cla4. After membrane extraction mediated by Rdi1, Rho4 is degraded by a novel mechanism, which includes the glycogen synthase kinase 3␤ homologue Ygk3, vacuolar proteases, and the proteasome. Together, these results indicate that Rdi1 uses distinct modes of regulation for different Rho GTPases. INTRODUCTIONSmall guanosine triphosphatases (GTPases) of the Rho family control fundamental processes of cell biology common to all eukaryotes, such as morphogenesis, polarity, movement, and cell division (Jaffe and Hall, 2005). In the budding yeast Saccharomyces cerevisiae, which encodes six Rho GTPases (Cdc42 and Rho1 to Rho5), these proteins play a pivotal role in the establishment of cell polarity (Park and Bi, 2007). Budding yeast cells undergo polarized growth during various phases of their life cycle, including budding during vegetative growth, mating between haploid cells of opposite mating types, and filamentous growth upon nutrient limitation. Although Cdc42 and Rho1 are well characterized, little is known about the other Rho proteins. Membrane association of Rho-type GTPases is essential for their function, and it depends on a C-terminal prenyl moiety and an adjacent polybasic region. Cdc42 localizes to internal membranes and the entire plasma membrane, where it clusters at sites of polarized growth, including the tips of mating projections, incipient bud sites, the tips and sides of growing buds, and the bud neck region of large-budded cells (Ziman et al., 1993;Richman et al., 2002). In addition to membranebound Cdc42, a cytoplasmic pool has been found (Ziman et al., 1993). Similar to Cdc42, Rho1 has been shown to localize at sites of polarized growth .Like other regulatory GTPases, they act as molecular switches, cycling between an active guanosine triphosphate (GTP)-bound state and an inactive guanosine diphosphate (GDP)-bound state. This activity is highly regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). The exchange of GDP to GTP, and thus the activation of Cdc42, is catalyzed by GEFs. In the active GTP-bound state, Rho GTPases perform their regulatory function through a conformation-specific interaction with effector proteins. G...

Membrane Organization and Dynamics in Cell Polarity

“…In the fungus Cryptococcus neoformans, Cdc42 is mislocalized to the cytoplasm when the Rho GDI homolog is overexpressed (Price et al 2008). This is consistent with published data that recombinant Rdi1 or overexpressed Rdi1 in yeast can extract Rho proteins from the plasma membrane or vacuole membrane (Eitzen et al 2001;Tcheperegine et al 2005;Tiedje et al 2008). Therefore, recycling through GDIs may, like endocytosis, play an important role in the establishment and maintenance of polarity.…”

Rga2 is a Rho2 GAP that regulates morphogenesis and cell integrity in S. pombe

“…Furthermore, we had observed a decrease in the total Rho2 level of cells lacking Rga2, Rga7 and Rga6, suggesting that GTP-Rho2 is also regulated by degradation. Some examples of Rho regulation by protein degradation have been described in animal cells (Wang et al, 2003;Schwamborn et al, 2007), and it has been recently published that Rdi, the only S. cerevisiae Rho GDI, regulates Rho4 degradation by a proteolytic pathway that includes the proteasome, vacuolar proteases and the GSK-3b homologue Ygk3 (Tiedje et al, 2008). Additional experiments would be required to study the hypothethical Rho2 regulation by degradation.…”

Morphogenesis and the Cell Cycle

“…Yeast contain a single Rho-GDI homolog, Rdi1p, that can extract prenylated Cdc42p from the membrane (Masuda et al 1994;Koch et al 1997;Tcheperegine et al 2005;Tiedje et al 2008), and work on the human Cdc42p/GDI interaction suggests that GDI preferentially extracts GDPbound (as opposed to GTP-bound) Cdc42p from membranes ). Because the cytoplasmic diffusion of Cdc42p-GDI complexes is expected to be fast and the yeast cell is small, the GDI could in principle "move" GDP-Cdc42p between outlying areas and the polarization site much faster than the rate at which GTP-Cdc42p diffuses at the plasma membrane ( Figure 3B).…”

“…Some studies reported the presence of Cdc42p in cytoplasmic fractions even in rdi1D mutant cells (Koch et al 1997;Tiedje et al 2008), suggesting that there are other mechanisms that can extract prenylated Cdc42p from membranes. If such (currently undescribed) mechanisms were selective for GDP-Cdc42p, then (like the GDI) they too would promote Cdc42p concentration at the polarization site.…”