Molecular studies of RNA-ER cotransport

Since localization studies have so far been focussed on ASH1 mRNA, we extended our analysis and studied the localization pattern of different localized mRNAs in wild type and aux1 cells at different cell cycle stages. In wild type cells we could show that WSC2, IST2, EAR1, MID2 and SRL1 are localized to 86-100% to the bud tip at early stages (S phase) and to 76-100% at late cell cycle stages (G2 and M phase). Deletion of AUX1 significantly inhibited localization of EAR1, MID2, IST2, SRl1 and WSC2 at S phase but more than 80% of aux1 cells could localize these mRNAs at later cell cycle stages. In contrast to previous studies, we did not see a dependence of ASH1 localization on AUX1 function, which might be related to the M-phase specific expression of ASH1. Previous studies showed that a localized mRNP, of which the RNA-binding protein She2p is a component of, binds to the tip of ER-membrane tubules (Schmid et al, 2006). In vitro, She2p interacts with purified ER membranes even after protease treatment of these membranes. It has also been shown that it binds to artificial liposomes not containing any proteins (M. Schmid, unpubl.). Since lipid composition of these liposomes did not alter the binding, we investigated wheter membrane curvature influences binding, i.e. if liposomes of different size show of differences in binding capacity for She2p. Additionally, we investigated if binding of She2p to the ER membranes is saturable, indicating a specific binding partner. Results of these studies will be presented.