Among the three functionally characterized vesicles within the cell (Clathrin, COPII and COPI coated vesicles), COPI vesicles mediate the transport in both anterograde and retrograde transport within the Golgi complex (Orci et al., 1997) as well as recycling of proteins from the Golgi to the ER (Cosson and Letourneur, 1994; Letourneur et al., 1994). An in vitro based assay using soluble coatomer and ARF1 together with synthetic liposomes containing p23 tail peptide yielded generation of COPI vesicles, thus establishing the minimal requirements for COPI coat assembly (Bremser et al., 1999). In a program to reconstitute one round of budding, uncoating and fusion of a COPI vesicle from defined components, the next step is to include in a liposomal system the components needed for the fusion reaction. For that purpose, SNAREs (Soluble N-Ethylmaleimid-sensitive fusion protein Attachment protein REceptors) were required since they were shown to be the machinery for fusion (McNew et al., 2000; Nickel et al., 1999; Sollner et al., 1993; Weber et al., 1998)). To study their behavior in the COPI budding process, different ER and Golgi SNAREs (Sec22p, Vti1p, Gos1p, Bos1p, Bet1p, Ykt6p) were produced in bacteria, purified and reconstituted into liposomes in their correct physiological orientation. Sec22p and Vti1p do not seem to be preferentially taken up in COPI coated vesicles under the conditions of the in vitro budding assay. Preliminary data allowed reconstitution of SNARE complexes and further experiments should allow the study of the mechanisms involved in their specific uptake in COPI vesicles. Prior to fusion, vesicles need to be uncoated. This process was shown to be dependent on ARF1-GTP hydrolysis (Tanigawa et al., 1993), a reaction catalyzed by ARF-GTPase activating protein (ARF-GAP). Therefore myristoylated yeast ARF1p as well as its ARF-GAP (Glo3p) was produced in bacteria, purified to apparent homogeneity and in an active state with regard to exchange of nucleotide and GTP hydrolysis in presence of liposomes. Moreover, selecting for large size liposomes used in the in vitro budding assay was critical to ensure newly formed vesicles are authentic ones and not preexisting small structures. Therefore, gel filtration experiments were successfully used to achieve this goal. Tools were provided to reconstitute one round of vesicular transport in vitro. Active proteins (ARF1p, coatomer) involved in coat assembly, ARF-GAP required for uncoating and the fusion machinery proteins SNAREs were provided. A Homogenous population of large liposomes was generated so that the total signal observed after budding is due only to de novo generated COPI vesicles and not to preexisting small structures.
|Supervisor:||Dr. Felix T. Wieland, Prof.|
|Date of thesis defense:||10 September 2003|
|Faculties / Institutes:||Service facilities > Heidelberg University Biochemistry Center|
|Subjects:||570 Life sciences|