Yeast formin Bni1p has multiple localization regions that function in polarized growth and spindle orientation

Abstract

Formins are conserved proteins that assemble unbranched actin filaments in a regulated, localized manner. Budding yeast's two formins, Bni1p and Bnr1p, assemble actin cables necessary for polarized cell growth and organelle segregation. Here we define four regions in Bni1p that contribute to its localization to the bud and at the bud neck. The first (residues 1-333) requires dimerization for its localization and encompasses the Rho-binding domain. The second (residues 334-821) covers the Diaphanous inhibitory-dimerization-coiled coil domains, and the third is the Spa2p-binding domain. The fourth region encompasses the formin homology 1-formin homology 2-COOH region of the protein. These four regions can each localize to the bud cortex and bud neck at the right stage of the cell cycle independent of both F-actin and endogenous Bni1p. The first three regions contribute cumulatively to the proper localization of Bni1p, as revealed by the effects of progressive loss of these regions on the actin cytoskeleton and fidelity of spindle orientation. The fourth region contributes to the localization of Bni1p in tiny budded cells. Expression of mislocalized Bni1p constructs has a dominant-negative effect on both growth and nuclear segregation due to mislocalized actin assembly. These results define an unexpected complexity in the mechanism of formin localization and function.

FIGURE 1:

The N-terminal region of Bni1p has three localization domains. (A) Schematic diagram of Bni1p domains based on homology to mDia1, and the constructs examined. Constructs that localized to the bud cortex and bud neck during the right stages of the cell cycle are shown in green, whereas constructs that did not localize are shown in red. CC, predicted coiled coil; DAD, Diaphanous-autoinhibitory domain; DID, Diaphanous inhibitory domain; DD, dimerization domain; FH1, formin homology domain 1; FH2, formin homology domain 2; GBD, GTPase-binding domain; SBD, Spa2p-binding domain. (B–O) GFP images of cells overexpressing Bni1p N-terminal constructs in a bni1Δ background. Bni1p constructs were tagged with GFP at the C-terminus with expression from the GAL1 promoter on a CEN plasmid. Cells were examined after 3–4 h induction with galactose. (M–O) GFP images of cells overexpressing the Bni1p N-terminal regions in a bni1Δ background after treatment with 200 μM latrunculin A for 10 min. Scale bar, 5 μm.

FIGURE 2:

Deletion of N-terminal regions of Bni1p leads to progressive defects in growth and polarity. (A) Schematic diagram of BNI1 truncation constructs, with the two phenotypic classes indicated. (B) Growth of 10-fold serial dilutions of cells with N-terminal deletions in a BNR1 and bnr1Δ background grown at 26°C on yeast extract/peptone/dextrose (YPD) plates. (C) F-Actin staining with phalloidin and GFP localization of the indicated constructs in BNR1 cells. Scale bars, 5 μm. (D) Immunoblot with antibodies against GFP to show expression of the indicated Bni1p constructs.

FIGURE 3:

Spa2p contributes to cell growth both through binding the SBD of Bni1p and independent of the SBD. (A) Growth assays of class I Bni1p truncations in SPA2 and spa2Δ cells at 26°C on YPD plates. (B) F-Actin staining with phalloidin and GFP localization of the indicated constructs in spa2Δ cells. Scale bar, 2 μm. (C) Growth of spores derived from individual tetrads (arrayed vertically) after sporulation of the indicated diploids. Circles indicate bni1Δ79-988 or bni1Δ79-1230 SPA2 spores; rectangles indicate bni1Δ79-988 or bni1Δ79-1230 spa2Δ spores.

FIGURE 4:

Deletion of N-terminal regions of Bni1p leads to progressive defects in nuclear segregation. (A) Growth of 10-fold serial dilutions of cells with N-terminal deletions in ARP1 (SC-URA plates that retain the CEN-URA3-ARP1 plasmid) and arp1Δ (5-fluoroorotic acid plates that select against the CEN-URA3-ARP1 plasmid) cells grown at 26°C. (B) Growth of 10-fold serial dilutions of cells with class I N-terminal deletions in ARP1 and arp1Δ cells at either at 26 or 14°C on YPD plates or at 26°C in the presence of 10 μg/ml benomyl. (C) Percentage of class I deletions in ARP1 and arp1Δ cells with the indicated number of nuclei in the mother cell. Cells were grown at room temperature until OD = 0.2 and then shifted to 16°C for 8 h, and >500 cells were analyzed following staining with 4′,6-diamidino-2-phenylindole. (D) Percentage of cells with misoriented spindles in small- to medium-budded cells. n > 100 for each sample. (E, F) Growth of 10-fold serial dilutions of cells as indicated under the same conditions used in B.

FIGURE 5:

The dominant effects of a class II Bni1p deletion are due to mislocalized assembly of actin. (A) Growth of spores derived from individual tetrads (arrayed vertically) after sporulation of the indicated diploids. Circles indicate BNI1 arp1Δ spores; rectangles indicate bni1Δ79-998 arp1Δ or bni1Δ79-998-I1431A arp1Δ spores. (B) Growth of 10-fold serial dilutions of cells with the indicated genotypes on YPD plates at 26 and 14°C. (C) Growth of cells expressing bni1Δ79-988 without or with an appended N-terminal CRIB domain. (D) F-Actin staining with phalloidin and GFP localization of the indicated construct. Scale bars, 5 μm.