She3p Possesses a Novel Activity Required for ASH1 mRNA Localization in Saccharomyces cerevisiae

Abstract

Intracellular and intercellular polarity requires that specific proteins be sorted to discreet locations within and between cells. One mechanism for sorting proteins is through RNA localization. In Saccharomyces cerevisiae, ASH1 mRNA localizes to the distal tip of the bud, resulting in the asymmetric sorting of the transcriptional repressor Ash1p. ASH1 mRNA localization requires four cis-acting localization elements and the trans-acting factors Myo4p, She3p, and She2p. Myo4p is a type V myosin motor that functions to directly transport ASH1 mRNA to the bud. She2p is an RNA-binding protein that directly interacts with the ASH1 mRNA cis-acting elements. Currently, the role for She3p in ASH1 mRNA localization is as an adaptor protein, since it can simultaneously associate with Myo4p and She2p. Here, we present data for two novel mutants of She3p, S348E and the double mutant S343E S361E, that are defective for ASH1 mRNA localization, and yet both of these mutants retain the ability to associate with Myo4p and She2p. These observations suggest that She3p possesses a novel activity required for ASH1 mRNA localization, and our data imply that this function is related to the ability of She3p to associate with ASH1 mRNA. Interestingly, we determined that She3p is phosphorylated, and global mass spectrometry approaches have determined that Ser 343, 348, and 361 are sites of phosphorylation, suggesting that the novel function for She3p could be negatively regulated by phosphorylation. The present study reveals that the current accepted model for ASH1 mRNA localization does not fully account for the function of She3p in ASH1 mRNA localization.RNA localization is a process to spatially and temporally restrict intracellular and intercellular protein expression. This mechanism for protein sorting is utilized by a number of eukaryotic organisms including mammals, Drosophila melanogaster, Xenopus spp., and Saccharomyces cerevisiae (4,15,25,37,44). RNA localization can be achieved through at least three distinct mechanisms: cytoplasmic diffusion followed by entrapment of the RNA at the site of localization, generalized degradation/localized protection, and directed transport using motor proteins and cytoskeletal elements (60). S. cerevisiae utilizes directed transport to localize at least 30 mRNAs to the bud tip (2,3,57,61). Localization of ASH1 mRNA to the bud tip in yeast results in the asymmetric sorting of Ash1p to the daughter cell nucleus (10,43,62). Ash1p is a transcriptional repressor that prevents expression of the HO endonuclease in the daughter cell, restricting mating-type switching exclusively to the mother cell (6,36,45,58).ASH1 mRNA localization is dependent on She2p, She3p, and Myo4p (43, 62). She2p is an RNA-binding protein that associates with the four ASH1 mRNA cis-acting localization elements: E1, E2A, E2B, and E3 (7,11,26,41,48). She2p also directly associates with the C terminus of She3p (7,11,26,41,48). The N terminus of She3p has the ability to associate with the type V myosin, Myo4p (7,19,47). Furthermore...

A Cytoplasmic Complex Mediates Specific mRNA Recognition and Localization in Yeast

“…Although previous studies did not hint at a nuclear role of She3p [17],[18],[21]–[23],[36], this has not been tested directly. It is therefore a remaining possibility that the ternary complex also plays a functional role in the nucleus.…”

“…Previous studies suggested that She3p acts only in the cytoplasm [17],[18],[21]–[23],[36]. However, this assumption had not been rigorously tested before.…”

Structure of a myosin•adaptor complex and pairing by cargo

“…She3p's binding site for Myo4p has been localized to the middle region of She3p (30), with a genetically determined upstream binding site for tER and a downstream binding site for zipcoded mRNP (29). Recently, the C-terminal region of She3p has also been reported to contain an RNA-binding site (25,31).…”

Hooking She3p onto She2p for myosin-mediated cytoplasmic mRNA transport

“…Instead, cargo binds via an "adaptor" protein, swi5p-dependent HO expression 3 protein (She3p) (7). Notably, the 425-residue-long She3p contains at least four binding modules that appear to be sequentially arranged along its primary structure (8)(9)(10)(11). Our recent composite crystal structure of a region of She3p (comprising residues showed that two She3p molecules form a pseudocoiled coil (6).…”