Browsing by Author "Wicky John, Sidonie"

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    Characterization of the yeast proteins Neo1p and Sjl2p, two highly conserved regulators of phospholipid composition within endosomal membranes
    (2004) Wicky John, Sidonie; Singer-Krüger, Birgit (Priv.-Doz. Dr.)
    Endocytosis is a key membrane trafficking pathway by which all eukaryotic cells internalize extracellular material as well as portions of the plasma membrane. In the budding yeast Saccharomyces cerevisiae, the major fraction of the material internalized at the plasma membrane is transported through the early and late endosomal compartments to the vacuole. The endosomal compartments are highly dynamic and are connected to the plasma membrane, the late Golgi complex and the vacuole by several routes, which all involve vesicular transport. Thus, the formation of vesicles is essential to maintain the dynamic exchanges between these different compartments. In the present study, I analyzed two yeast proteins, Neo1p and Sjl2p, which are suggested to function as regulators of the phospholipid composition of endosomal organelles and thereby seem to participate in vesicle formation (Neo1p) and in vesicle uncoating (Sjl2p), two processes essential during vesicular membrane transport. Neo1p is an essential member of the Drs2 family of P-type ATPases with proposed function as aminophospholipid (APL) translocases. Genetic and biochemical data in the laboratory of B. Singer-Krüger indicated that Neo1p might function within the endosomal system. Consistent with these findings, I could confirm by indirect immunofluorescence that the major fraction of Neo1p localizes to the endosomal compartments, while a smaller part associates with late Golgi membranes. In agreement with this localization, two temperature-sensitive neo1 mutants were shown to be defective in endocytosis (B. Singer-Krüger's laboratory) and in vacuolar protein sorting (my studies). While these defects were already observed at permissive temperature, at nonpermissive temperature the neo1-ts mutants exhibited additional impairments in membrane transport between the ER and the early Golgi compartment. However, these defects were most likely a consequence of the accumulation of mutant Neo1 proteins in the ER under these conditions. In support of previous results in the laboratory of B. Singer-Krüger, I identified further links between Neo1p and the endosomal proteins Ysl2p and Arl1p. I could show that Neo1p interacts in vivo with Ysl2p and that the subcellular localization and the stability of Ysl2p are affected in the neo1-69 mutant. Furthermore, the subcellular distribution of Arl1p was also found to be impaired in neo1-69 cells at permissive temperature. Based on these findings and the work of B. Singer-Krüger, Neo1p was proposed to act together with Ysl2p and Arl1p in membrane trafficking within the endosomal/late Golgi system. In the second part of this work, the subcellular localization of Sjl2p, a polyphosphoinositide- and 5'-inositide phosphatase of the synaptojanin family, was examined. Based on genetic analyses, Sjl2p has been suggested to participate in early steps of endocytic transport. Here, I determined by indirect immunofluorescence that the Sjl2p-positive punctate structures were distinct from those containing typical early and late endosomal marker proteins and were not sensitive to mutations affecting the structure of either the endosomal compartments or the Golgi complex. Sjl2p did not show a typical plasma membrane staining pattern either. However, Sjl2p colocalized with cortical actin patch components found within clumps that accumulated in cells lacking the two actin-regulating kinases Prk1p and Ark1p. Within these aberrant structures, Sjl2p also colocalized with FM4-64 endocytosed for a short time, suggesting that Sjl2p localized to primary endocytic vesicles that interact with the cortical actin cytoskeleton. This interaction may at least to some extent be mediated by Bsp1p, a binding partner of Sjl2p isolated in B. Singer-Krüger's laboratory, which in my studies was found to be part of the cortical actin cytoskeleton. In cells deficient for the PtdIns(4)P kinase (pik1-ts), the staining pattern of Bsp1p was changed, suggesting that the subcellular distribution of Bsp1p is dependent on the levels of phosphoinositides. Consistent with that, Bsp1p was found to flotate with liposomes containing acidic phospholipids including phosphoinositides. Thus, Bsp1p may act as an adapter that directly connects Sjl2p-containing vesicles to the cortical actin cytoskeleton during early stages of endocytosis via interactions with Sjl2p (B. Singer-Krüger) and with a subset of phospholipids within the vesicle membrane (B. Singer-Krüger and my results). In summary, the results of my PhD thesis brought new insights into the localization and function of Neo1p and Sjl2p within the endosomal system. These data are relevant for future studies to elucidate the precise mechanism of Neo1p and Sjl2p during endocytosis.