Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with Myo2 and Exo70 - PubMed (original) (raw)

Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with Myo2 and Exo70

N G Robinson et al. Mol Cell Biol. 1999 May.

Abstract

The Rho3 protein plays a critical role in the budding yeast Saccharomyces cerevisiae by directing proper cell growth. Rho3 appears to influence cell growth by regulating polarized secretion and the actin cytoskeleton, since rho3 mutants exhibit large rounded cells with an aberrant actin cytoskeleton. To gain insights into how Rho3 influences these events, we have carried out a yeast two-hybrid screen using an S. cerevisiae cDNA library to identify proteins interacting with Rho3. Two proteins, Exo70 and Myo2, were identified in this screen. Interactions with these two proteins are greatly reduced or abolished when mutations are introduced into the Rho3 effector domain. In addition, a type of mutation known to produce dominant negative mutants of Rho proteins abolished the interaction with both of these proteins. In contrast, Rho3 did not interact with protein kinase C (Pkc1), an effector of another Rho family protein, Rho1, nor did Rho1 interact with Exo70 or Myo2. Rho3 did interact with Bni1, another effector of Rho1, but less efficiently than with Rho1. The interaction between Rho3 and Exo70 and between Rho3 and Myo2 was also demonstrated with purified proteins. The interaction between Exo70 and Rho3 in vitro was dependent on the presence of GTP, since Rho3 complexed with guanosine 5'-O-(3-thiotriphosphate) interacted more efficiently with Exo70 than Rho3 complexed with guanosine 5'-O-(3-thiodiphosphate). Overlapping subcellular localization of the Rho3 and Exo70 proteins was demonstrated by indirect immunofluorescence. In addition, patterns of localization of both Exo70 and Rho3 were altered when a dominant active allele of RHO3, RHO3(E129,A131), which causes a morphological abnormality, was expressed. These results provide a direct molecular basis for the action of Rho3 on exocytosis and the actin cytoskeleton.

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Figures

FIG. 1

FIG. 1

Fragments of Exo70 and Myo2 which interact with Rho3. Regions of Exo70 and Myo2 contained in the two-hybrid clones identified to interact with Rho3 are shown as bars below the intact molecules. ATP, ATP binding region; AB, actin binding region; N, neck region; C, coiled-coil region; H, hinge region.

FIG. 2

FIG. 2

The yeast two-hybrid interaction between both Rho3 and Rho1 with their downstream effectors. Rho3 and Rho1 were fused to the DNA binding domain of GAL4. Their interactions with Pkc1, RBP4, RBP26, and Bni1, all of which are fused to the activation domain of GAL4, are demonstrated by the plate β-galactosidase assay.

FIG. 3

FIG. 3

Complementation of a Δ_rho3_ yeast with various RHO3 mutants. Yeast strain YMR504 was transformed with RHO3, rho3V25, rho3A48, rho3S47, and rho3N30, as well as RHO1, all of which were inserted into the centromeric vector pRS316. Transformants were grown on medium containing either galactose (A) or glucose (B) and assessed for the ability to complement the growth defect exhibited by Δ_rho3_ cells. wt, wild type.

FIG. 4

FIG. 4

In vitro interaction of Rho3 with RBP26, Exo70, and RBP4. (A) GST-Rho3 preincubated with a nonhydrolyzable analogue of GDP or GTP was mixed with either MBP-RBP4 or MBP-Exo70 bound to amylose resin. After rinsing, the resin was collected, loading buffer was added, and the samples were boiled. The proteins were then separated on an SDS-polyacrylamide gel, and the presence of GST-Rho3 was examined by using an anti-GST antibody. The Rho3 lane contains purified GST-Rho3 and is intended to show the position of GST-Rho3. (B) GST-Kir (20 μg), GST-Rad (20 μg), GST (20 μg), GST–H-Ras (20 μg), GST-Rho4 (10 and 20 μg), and GST-Rho3 (10 and 20 μg) were loaded with GTPγS and assessed for the ability to bind MBP-Exo70 as described above. The Rho3 lane contains purified GST-Rho3 as in panel A. (C) GST-Rho3 and GST-Rho3A48 were loaded with GTPγS and assessed for the ability to bind MBP-RBP4 as described above. The Rho3wt (wild-type Rho3) and Rho3A48 lanes contain purified Rho3 proteins. (D) GST-Rho3 was loaded with GTPγS and assessed for its ability to bind to either MBP, MBP-RBP26, or MBP-RBP4 as described above. The Rho3 lane contains purified GST-Rho3 as in panel A.

FIG. 5

FIG. 5

Localization of Exo70 and Rho3. (A) HA-Exo70-producing cells carrying GAL7p:RHO3E129 were cultured in SCGal-U at 30°C. (B) HA-Exo70-producing cells carrying GAL7p:RHO3E129,A131 growing exponentially in SCGal-U at 30°C were shifted to 15°C and harvested 48 h after the shift. Cells were fixed and stained with anti-HA (middle) and anti-Rho3 (right) antibodies. Phase photographs are also shown (left).

FIG. 6

FIG. 6

Schematic showing the various effectors of Rho3 and the cellular processes that they regulate. See Discussion for an explanation of each interaction.

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