%0 Generic
%A Jaedicke, Andreas Martin
%D 2004
%F heidok:4600
%K mRNS Lokalisation , Translationskontrolle , RNP Komplextandem affinity purification , ASH1 mRNA
%R 10.11588/heidok.00004600
%T Biochemical purification and functional characterization of the She RNP complex from S. cerevisiae
%U https://archiv.ub.uni-heidelberg.de/volltextserver/4600/
%X Asymmetric mRNA localization is a widely used mechanism to sort cell fate determinants in development.  In the budding yeast Saccharomyces cerevisae ASH1 (for 'asymmetric synthesis of HO') mRNA localization to the tip of the growing bud leads to targeting of Ash1p to the daughter cell nucleus prior to cytokinesis and is a prerequisite for proper mating type switching. In a genetic screen 5 SHE (for 'Swi5p-dependent HO expression') genes have been isolated, coding for proteins required for the RNA localization process.  SHE1 is equivalent to MYO4, a locus that encodes a member of the class V unconventional myosins. The finding that a motor protein is required for ASH1 mRNA targeting suggested a cytoskeleton-based, active transport mechanism. Functional characterization of the SHE genes in turn established a working model for the 'core She machinery' which implies She1p / Myo4p as the ATP-dependent motor protein, She2p as the ASH1 mRNA-binding protein, and She3p as adapter protein connecting She2p to She1p / Myo4p.  She4p has been suggested to function in myosin assembly, whereas She5p is required for cell cycle regulated remodeling of the actin cytoskeleton. In a shared project with C. Kruse we could show that Myo4p trafficking is regulated by the formation of a robust She RNP, and relies on She2p and RNA association. In addition to the She proteins, accessory factors such as Loc1p or Khd1p have been suggested to function in ASH1 mRNA localization though they have not been identified in the genetic screen. Thus, in order to allow a detailed characterization of the ASH1-She RNP ('ribo-nucleoprotein') complex, I initiated a biochemical purification.  In order to enrich for the She RNP two myo4p mutants were generated in the myosin ATPase, a domain required for Myo4p force generation. Localization studies reveal that the mutants do not transport ASH1 mRNA anymore to the bud tip but instead accumulate in an intermediate, 'frozen' state in the cytoplasm.  Affinity purification was carried out based on the TAP ('tandem affinity purification') protocol, using two alternative bait proteins (She2-TAP or Myo4-TAP).  In either case I could purify the core She machinery together with ASH1 mRNA.  Further analysis of the She RNP by gel filtration experiments revealed a peak fraction with a molecular weight of approximately 4.5 MDa.  Within this fraction I could identify Myo4p, She2p and ASH1 mRNA, arguing for the integrity of a single RNP.  In addition to She1-3p mass-spectrometric analysis identified a number of so far unknown proteins, including the kinase Gin4p and the translation inhibitor Eap1p.  Eap1p has been of outstanding interest since a systematic RNA localization assay with ash1 mutants that contained premature stop codons inserted at various positions within the coding sequence have revealed severe localization defects, indicating that translation (and translational regulation) is required for correct localization. Eap1p has been characterized as an inhibitor of translation initiation and belongs to the family of 'eukaryotic initiation factor 4E-binding proteins', eIF 4E-BPs.  Members of this protein family share a common sequence motif which mediates association with eIF 4E, thereby blocking initiation of translation.  Initial studies with D eap1 yeast strains have revealed a defective partial accumulation of Ash1p in mother cell nuclei, whereas ASH1 mRNA localization to the bud tip and total Ash1p levels remained unaffected.  This observation prompted me to introduce a new model for ASH1 mRNA localization, including the regulation of translation initiation during cytoplasmic She RNP trafficking to the bud.