The cell polarity proteins Boi1 and Boi2 direct an actin nucleation complex to sites of exocytosis in Saccharomyces cerevisiae

Abstract

Due to the local enrichment of factors that influence its dynamics, and organization, the actin cytoskeleton displays different shapes and functions within the same cell. In yeast cells post-Golgi vesicles ride on long actin cables to the bud tip. The proteins Boi1 and Boi2 participate in tethering and docking these vesicles to the plasma membrane. Here we show that Boi1/2 also recruit nucleation and elongation factors to form actin filaments at sites of exocytosis. Disrupting the connection between Boi1/2 and the nucleation factor Bud6 impairs filament formation, reduces the directed movement of the vesicles to the tip, and shortens their tethering time at the cortex. Transplanting Boi1 from the bud tip to the peroxisomal membrane partially redirects the actin cytoskeleton and the vesicular flow towards the peroxisome, and creates an alternative, rudimentary vesicle-docking zone. We conclude that Boi1/2 through their interactions with Bud6 and Bni1 induce the formation of a cortical actin structure that receives and aligns incoming vesicles before fusion with the membrane.

Regulation of intrinsic polarity establishment by a differentiation-type MAPK pathway in S. cerevisiae

“…Therefore, proteins that spatially promote budding promote fMAPK activity, and proteins that spatially restrict budding also restrict fMAPK pathway activity. Cells lacking other Cdc42p-interacting proteins, Boi1p and Boi2p (Bender et al, 1996;Glomb et al, 2020;Liao et al, 2013;Masgrau et al, 2017) and Msb1p (Bender and Pringle, 1991;Bi et al, 2000;Liao et al, 2013) were examined but did not show altered activity of fMAPK pathway or a difference in invasive growth compared to wild-type cells ( Fig. S4D-I).…”

Type V Myosin focuses the polarisome and shapes the tip of yeast cells

“…Furthermore, Myo2 bound to Pea2 should retain the full ability to walk along actin cables. To test our hypothesis, we resorted to a Pex3-Pea2 fusion construct that displayed Pea2 on the surface of peroxisomes (Pea2peroxisomes) ( Figure 4A) (Luo et al, 2014;Glomb et al, 2020). If our three assumptions are correct, the cellular localization of Pea2-peroxisomes should be altered by a Myo2-dependent transport along the actin cables.…”

“…Yeast strains were cultivated in SD or YPD media at the indicated temperatures. Media preparation followed standard protocols (Glomb et al, 2020). SD medium for Split-Ub assays contained in addition 1 mg/ml 5-fluoro-orotic acid (5-FOA, Formedium, Hunstanton, Norfolk, UK).…”

“…GFP-Sec4 and PBD Gic2 -RFP were expressed in yeast by genomic integration of plasmids pSec4prom-Sec4-GFP306, or YIp211-GIC2-PBD-1.5tdTomato into the ura3-52 locus (Glomb et al, 2020). myo2 R1419A (myo2 RA ), myo2 R1419D (myo2 RD ) and…”

“…MYO2 WT alleles were obtained by recombination of PCR fragments generated from plasmids pFA-Myo2-CBD(R1419A)-hph, pFA-Myo2-CBD(R1419D)-hph, or pFA-Myo2-CBD(WT)-hph using a forward primer starting at amino acid1152 and a standard reverse S2 primer annealing to the MYO2 terminator. The chimeric pP MET -Pex3 1-45 -mCherry 306 plasmid was adapted from Luo et al, 2014(Luo et al, 2014Glomb et al, 2020). PEA2 was amplified from genomic DNA and inserted in frame behind the mCherry tag using BamHI/SalI restriction sites.…”