Browsing by Author "Böttcher-Sehlmeyer, Claudia"

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    Subcellular localization and molecular interactions of phosphoinositide 5'-phosphatases of the yeast synaptojanin-like protein family
    (2006) Böttcher-Sehlmeyer, Claudia; Singer-Krüger, Birgit (PD Dr.)
    Phosphorylated derivates of phosphatidylinositol, collectively called phosphoinositides (PIs), are a minor class of short-lived lipids that control numerous cellular processes including cell signaling, growth, vesicular traffic, and actin cytoskeletal arrangements. Different PI isoforms are concentrated in distinct subcellular compartments, with for example phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at the plasma membrane and PtdIns(4)P on Golgi membranes. PIs also play a role in the mechanism by which the direction of membrane traffic is controlled. To guarantee the proper vesicle formation and targeting to the acceptor compartment, the differential distribution of PIs is maintained by specific PI kinases, which localize to specified subcellular compartments. Conversely, a set of phosphatases and lipases regulates the PI turnover. Previous studies implicated de-phosphorylation of PtdIns(4,5)P2 by the mammalian polyphosphoinositide phosphatase synaptojanin 1 as an important step in endocytosis. Since then, indirect evidence exists in different model organism for a role of the synaptojanin proteins in the un-coating of clathrin-coated endocytic vesicles. Members of the highly conserved synaptojanin family are defined by a three-domain structure. The N-terminal domain, homologous to yeast Sac1p, exhibits in some family members polyphosphoinositide phosphatase (PPIP) activity that can hydrolyse the phosphate at the 3', 4', or 5' position of the inositol ring. The central PI 5'phosphatase (5-Pase) domain specifically hydrolyses the phosphate at the 5' position of PtdIns(4,5)P2. The C-terminal proline-rich domain is more variable among the different family members. The yeast Saccharomyces cerevisiae contains three synaptojanin-like (Sjl) proteins, Sjl1p, Sjl2p, and Sjl3p. All three family members exhibit 5-Pase activity, while only Sjl2p and Sjl3p possess the PPIP activity. While previously Sjl1p has not been assigned to vesicular transport, Sjl3p has been implicated in clathrin-mediated membrane traffic between the TGN and endosomes. Since less data existed about the function of Sjl2p, the work of this thesis focused on further evidence for a specific role of Sjl2p in clathrin-mediated endocytosis. Initially, in this PhD thesis evidence was provided for an association of each family member with filamentous actin assembled in vitro. However, Sjl1p seems to be associated with F-actin more tightly than Sjl2p and Sjl3p. As already described above, earlier studies provided evidence for a localization and function of the three yeast synaptojanin family members at distinct subcellular compartments. To identify specific Sjl2p-binding partners, GST-pulldown assays and co-immunoprecipitation experiments were performed. GST-pulldown assays using the proline-rich domain of Sjl2p revealed the association of the following proteins: the putative endocytic vesicle scission factor Rvs167p, the early endocytic protein End3p, and, most importantly, clathrin heavy chain (Chc1p), the major component of clathrin-coated vesicles. Although most likely transiently, Sjl2p seems to be part of the Sla1p/Pan1p complex. Sla1p and Pan1p are multivalent proteins, which bind to a variety of endocytic proteins and thereby regulate the progress of early steps of endocytosis. In yeast, the question was still unresolved whether the synaptojanin-like proteins are associated with clathrin-coated vesicles (CCVs). Sephacryl S-1000 gel filtration chromatography resulted in co-fractionation of Sjl2p and Sjl3p with clathrin-coated vesicles, while Sjl1p eluted with the main peak of the cytoplasmic phosphoglycerate kinase and actin, well separated from vesicles. Although the immunodetection of Sjl2p with CCVs was unsuccessful by electron microscopy, several lines of evidence from this thesis and recently published work support the idea that Sjl2p resides on endocytic CCVs. Sjl3p is likely associated with CCVs derived from the TGN. An emerging body of evidence indicates that posttranslational modifications play a key role in the most, if not all, cellular processes in eukaryotes. The activity of a variety of plasma membrane transporters, channels, and receptors is regulated by internalization via the endocytic machinery. The internalization is initiated by phosphorylation and/or the attachment of an ubiquitin moiety to specific amino acid residues. In S. cerevisiae, phosphorylation of trans-acting proteins is one of the key factors in the actin dynamics at an endocytic vesicle. Furthermore, recent studies have shown that some trans-acting proteins of the endocytic machinery are also mono-ubiquitinated, such as Rvs167p. In this PhD thesis it was found that Sjl2p and Sjl3p are modified by tyrosine phosphorylation as well as by ubiquitination, while Sjl1p was not. It is tempting to speculate that these modifications regulate the phosphatase activities of Sjl2p and Sjl3p.