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Clathrin-coated pits on the plasma membrane invaginate into coated vesicles to internalize receptors and membrane. The clathrin adaptor epsin contains an aminoterminal ENTH domain that binds PI(4,5)P₂ and a carboxy-terminal domain that binds clathrin, and accessory proteins such as AP2. Here, we assessed how inter- and intramolecular factors affect the contribution of epsin to coated-pit function in living cells. We found Dictyostelium epsin was not required for global clathrin function, but plays an essential role in spore development. We demonstrated that clathrin, but not AP2, was critical for epsin to associate with clathrin-coated pits. We found that the carboxy-terminal region of epsin was essential, but not sufficient, for targeting epsin within clathrin-coated pits on the plasma membrane. In addition to targeting epsin to the membrane, the amino-terminal ENTH domain regulates the interaction between epsin and clathrin, an essential property that cannot be replaced by an alternate PI(4,5)P₂ binding domain. Moreover, the ENTH domain facilitates the functional interaction between clathrin and actin during late stages of endocytosis, possibly by regulating the activity of the adaptor Hip1r. Both the ability to bind PI(4,5)P₂ and another function mediated by residue T107 are critical for the activity of the ENTH domain. Our results support a model where the ENTH domain coordinates with the clathrin-binding C-terminal domain to allow a dynamic interaction of epsin with coated pits. Furthermore, we propose that the ENTH domain of epsin facilitates the membrane recruitment and phosphorylation of Hip1r, which in turn mediates the productive interaction of clathrin with the actin cytoskeleton at the plasma membrane. / text

The function of CABP1, a novel cAMP-binding protein in Dictyostelium discoideum, has been investigated. Previously, it has been shown that the molecular weight of CABP1 varies in different strains and that this variation is correlated with differences in response to cAMP. Sequence analysis showed that compared to its counterpart in strain AX2, the gene encoding CABP1 of strain V12M2 is missing 27 bp. AX2 transformants expressing the capA gene of V12M2 exhibited no change in phenotype, suggesting that CABP1 is not involved in the features examined or that the V12M2 variant is recessive. Disruption of capA by homologous recombination was attempted with no success, suggesting that this gene might be essential. Proteins interacting with CABP1 have been identified by immunoprecipitation using specific antibodies, CABP1 was also found to copurify with annexin VII, a calcium-dependent phospholipid-binding protein, suggesting that CABP1 and annexin VII might interact.

Universiẗat, Diss., 2006--Frankfurt (Main).

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Universiẗat, Diss., 2001--München.

Thesis (Ph. D.)--University of Wisconsin, 1967. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

University, Diss., 2004--Köln.

Univ., Diss., 2002--Frankfurt (Main)

The effects of stalk cell differentiation inducing factor (DIF) on stalk cell induction from vegetative cells and prestalk cells of Dictyostelium discoideum were investigated. It was found that the major DIF component DIF-1 is a poor inducer of stalk cell formation from prestalk cells, but a minor component, DIF-2 is a more important inducer for the conversion of prestalk to stalk cells. Evidence is presented that DIF-2 synergizes the activity of the other DIF components for prestalk to stalk cell conversion. In contrast DIF-5 inhibits the activity of DIF-1 and DIF-3/4. A model is proposed to explain those results. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate