The clathrin-coated pit is the major port of entry for many receptors and pathogens and is the paradigm for membrane-based sorting events in higher cells [1]. Recently, it has been possible to reconstitute in vitro the events leading to assembly, invagination, and budding off of clathrin-coated vesicles, allowing dissection of the machinery required for sequestration of receptors into these structures [2-6]. The AP2 adaptor complex is a key element of this machinery linking receptors to the coat lattice, and it has previously been reported that AP2 can be phosphorylated both in vitro and in vivo [7-10]. However, the physiological significance of this has never been established. Here, we show that phosphorylation of a single threonine residue (Thr156) of the mu2 subunit of the AP2 complex is essential for efficient endocytosis of transferrin both in an in vitro coated-pit budding assay and in living cells.
The formation of clathrin-coated pits at the plasma membrane requires the concerted action of many different molecules. The real challenge lies in determining the hierarchy of these interactions. We are using assays in both intact and permeabilized cells to dissect the temporal requirements for clathrin-coated vesicle formation, and also to examine the role of phosphorylation of the coat proteins.
We are interested in the roles that Src family protein tyrosine kinases play during the cell cycle in normal and transformed cells. Src family kinases are often activated as cells exit quiescence, and are frequently overexpressed and/or hyperactivated in human tumours, particularly of the breast and colon. Recently we have identified and characterized a small molecule that inhibits the ubiquitously expressed members of the Src family (Src, Fyn and Yes). Using this inhibitor (SU6656) we have confirmed that Src family kinases are required for platelet-derived growth factor (PDGF)-stimulated myc induction and mitogenic signalling, and begun to probe the 'Src pathway' in more detail. We have identified several proteins that are specific substrates of Src family kinases, and determined that Src kinases appear to antagonize the inhibitory effects of the tumour suppressor p53. Ir addition, we have been exploring the role of Src in the control of the cytoskeleton. The results of these studies will be presented.The type I1 restriction enzymes recognise specific D N A sequences and, in the presence of Mg(II), cleave both strands of the D N A at or adjacent to their recognition sites. Due to their value in D N A technology, screening programmes have yielded over 3000 such enzymes. It had been thought that these enzymes were rather similar to each other and could perhaps be categorised into two groups: the EcoRV-like and the EcoRI-like enzymes. The mechanism by which EcoRV find its target site in D N A will be shown here to be not by the generally accepted scheme of I-D 'sliding' but instead by 'hopping' and 'jumping' through 3-D space. Other type I1 enzymes will be shown to mediate long-range communications between distant sites, 'looping' out the intervening D N A .The cellular mechanisms of endocytosis are of fundamental importance, as this is the route by which essential macromolecules, but also some important infectious agents, access the cellular milieu. Receptors, loaded with cargo, are sequestered into coated pits and these pits form by the directed assembly of clathrin and the AP2 adaptor complex from the cytosol onto the membrane. Recently a plethora of other molecules, including dynamin, amphiphysin, endophilin and rab5 have been implicated in coated vesicle formation. A real challenge lies in determining the hierarchy of addition of these molecules and their mechanism of action. We have manipulated a permeabilised cell system to study the molecular requirements for individual stages in coated vesicle formation. A2The role of COP1 in vesicular transport. T. Nilsson nilsson@embl-heidelberg. de EMBL, CBB, Meyerhofstrasse 1, 0-69017, Heidelberg, GermanyIntra Golgi C O P I vesicles formed in vitro were shown to predominantly contain resident proteins of the Golgi stack. This included glycosylation enzymes and other resident proteins such as the family of small transmembrane proteins, the p24s. Only small amounts of anterograde cargo could be detected suggesting that the predominant role of COPI vesicles is to recycle ...
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