Rho1p-Bni1p-Spa2p interactions: implication in localization of Bni1p at the bud site and regulation of the actin cytoskeleton in Saccharomyces cerevisiae

Abstract

Rho1p is a yeast homolog of mammalian RhoA small GTP-binding protein. Rho1p is localized at the growth sites and required for bud formation. We have recently shown that Bni1p is a potential target of Rho1p and that Bni1p regulates reorganization of the actin cytoskeleton through interactions with profilin, an actin monomer-binding protein. Using the yeast two-hybrid screening system, we cloned a gene encoding a protein that interacted with Bni1p. This protein, Spa2p, was known to be localized at the bud tip and to be implicated in the establishment of cell polarity. The C-terminal 254 amino acid region of Spa2p, Spa2p(1213-1466), directly bound to a 162-amino acid region of Bni1p, Bni1p(826-987). Genetic analyses revealed that both the bni1 and spa2 mutations showed synthetic lethal interactions with mutations in the genes encoding components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p. Immunofluorescence microscopic analysis showed that Bni1p was localized at the bud tip in wild-type cells. However, in the spa2 mutant, Bni1p was not localized at the bud tip and instead localized diffusely in the cytoplasm. A mutant Bni1p, which lacked the Rho1p-binding region, also failed to be localized at the bud tip. These results indicate that both Rho1p and Spa2p are involved in the localization of Bni1p at the growth sites where Rho1p regulates reorganization of the actin cytoskeleton through Bni1p.

Figure 1

Bni1p–Spa2p interactions in the yeast two-hybrid method. Plasmids encoding AD_GAL4 and DBD_LexA fused to truncated Bni1ps were transformed into strain L40 expressing DBD_LexA-Spa2p(1213–1466) and AD_GAL4-Spa2p(1213–1466), respectively. Bni1ps-Spa2p(1213–1466) interactions were examined in each transformant by the qualitative and quantitative assay methods for β-galactosidase activity. Closed bars, blue colony color; open bars, white colony color. The values (Miller units) are the averages of β-galactosidase activities for three transformants. Each measured value was within 50% of the average. FH1 and FH2 are formin homology domains 1 and 2, respectively. (A) Interactions of Bni1ps with DBD_LexA-Spa2p(1213–1466). (B) Interactions of Bni1ps with AD_GAL4-Spa2p(1213–1466).

Figure 2

Direct Spa2p-Bni1p interaction. MBP-Spa2p(1213–1466) was incubated with either GST-Bni1p(826–987) or GST, coupled to glutathione Sepharose 4B beads. The beads were sedimented, the supernatants were saved, and the proteins bound to the beads were eluted. Comparable aliquots of the supernatants and the eluates were subjected to SDS-PAGE, followed by protein staining with Coomassie brilliant blue. Lanes 1 and 3, the supernatants; lanes 2 and 4, the eluates.

Figure 3

Cell morphology of the spa2 bck1 and bni1 bck1 mutants. Cells of haploid strains, OHNY2 (WT), DMYSU6 (spa2 bck1), and KFY5 (bni1 bck1), were incubated at 24°C in YPDAU medium for 10 h, fixed, and stained with rhodamine-phalloidin and DAPI for actin and DNA, respectively. Cells were subsequently subjected to microscopic observation. All fields were photographed at the same magnification. (A) The spa2 bck1 mutant. (B) The bni1 bck1 mutant.

Figure 4

Localization of Bni1p in the spa2 mutant. Cells of strains, BTY3 (SPA2) expressing myc-Bni1p, NTFSY1 (spa2) expressing myc-Bni1p, and OHNY1 (SPA2) expressing Bni1p, were incubated in SG-Trp medium for 8 h at 24°C, stained with the anti-myc monoclonal antibody, and subsequently subjected to immunofluorescence microscopic observation. (A) Localization of myc-Bni1p. All fields were photographed at the same magnification. (B) Percentages of small-budded cells with myc-Bni1p at the bud tip. More than 1000 cells with a small bud were observed for each determination.

Figure 5

Localization of Bni1p(490–1954), a Bni1p lacking the Rho1p-binding region. Cells of strains, BTY3 expressing myc-Bni1p or myc-Bni1p(490–1954) or OHNY1 expressing Bni1p, were incubated in SG-Trp medium for 8 h at 24°C, stained with the anti-myc monoclonal antibody, and subsequently subjected to immunofluorescence microscopic observation. (A) Localization of myc-Bni1p or myc-Bni1p(490–1954). All fields were photographed at the same magnification. (B) Percentages of small-budded cells with myc-Bni1p or myc-Bni1p(490–1954) at the bud tip. More than 1000 cells with a small bud were observed for each determination.