Abstract
Both the delivery of secretory vesicles and asymmetric distribution of mRNA to the bud are dependent upon the actin cytoskeleton in yeast. Here we examined whether components of the exocytic apparatus play a role in mRNA transport. By screening secretion mutants in situ and in vivo, we found that all had an altered pattern of ASH1 mRNA localization. These included alleles of CDC42 and RHO3 (cdc42-6 and rho3-V51) thought to regulate specifically the fusion of secretory vesicles but were found to affect strongly the cytoskeleton as well. Most interestingly, mutations in late secretion-related genes not directly involved in actin regulation also showed substantial alterations in ASH1 mRNA distribution. These included mutations in genes encoding components of the exocyst (SEC10 and SEC15), SNARE regulatory proteins (SEC1, SEC4, and SRO7), SNAREs (SEC9 and SSO1/2), and proteins involved in Golgi export (PIK1 and YPT31/32). Importantly, prominent defects in the actin cytoskeleton were observed in all of these strains, thus implicating a known causal relationship between the deregulation of actin and the inhibition of mRNA transport. Our novel observations suggest that vesicular transport regulates the actin cytoskeleton in yeast (and not just vice versa) leading to subsequent defects in mRNA transport and localization.
Highlights
The establishment of cell polarity in eukaryotes involves the asymmetric organization of mRNA, the cytoskeleton, and the secretory pathway to lead to the polarized distribution of new membrane along a given axis [1, 2]
ASH1 mRNA localization was examined in budded S and G2/M phase yeast cells by using Fluorescence in Situ Hybridization (FISH), and the results were statistically compiled from 30 to 80 cells per condition
A small number of cells (Ͻ20%) were found to have some mRNA located in the nucleus of the mother cell, the latter detected by propidium iodide staining, which probably represents a maternal pool of ASH1 mRNA that had yet to be exported and delivered to the daughter cell
Summary
MATa ade can his leu lys trp ura MATa his3⌬1 leu2⌬0 met15⌬0 ura3⌬0 MAT␣ his3-⌬ 200 leu2-3, ⌬112 lys2 ⌬801 trp901 suc2-⌬9 arf1⌬::HIS3 MATa leu cdc MATa his trp289 ura URA3::cdc42Ϫ6, GAL1p-CDC42::LEU2 MATa ade lys his trp leu ura cmd1-⌬1::TRP1 ade3::HIS3::cmd 1-239 MATa his3-⌬ leu2-3,-112 lys2-⌬801 200 trp901 suc2-⌬9 ura gga1::HIS5spL gga2::TRP1 1 MATa his leu trp ura myo MATa his3⌬1 leu2⌬0 met15⌬0 ura3⌬0 myo4D MATa leu2-3,-112 his3-⌬ 200 trp 901 lys2 ⌬801 suc2-⌬9 pik1⌬::HIS3 carrying pRS314pik. To a somewhat lesser extent, mutations in the Sec SNARE1 regulator [22], the exocyst components, Sec and Sec15 [23] (the latter being a possible Sec effector [24]), and the exocytic SNAREs, Sec, and Sso1/2 [25, 26], resulted in the mislocalization of ASH1 mRNA This suggests that SNAREs, SNARE regulatory, exocyst, and Golgi export proteins all exert control over mRNA transport and localization. Subsequent analysis of the actin cytoskeleton in these mutants revealed that all have a disorganized pattern of actin labeling, which occurs rapidly upon the shift to semi-restrictive and restrictive temperatures This indicates that a novel relationship exists between vesicle transport and the integrity of the actin cytoskeleton. Active exocytosis in yeast is necessary for both maintenance of the polarized actin cytoskeleton and subsequent mRNA transport and localization to the growing bud
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