TY  - THES
AB  - In the first project of this thesis possible interaction partners with the N-terminus of the Qb-SNARE Vti1p should be identified using the tandem affinity purification (TAP) method. Interaction partners were identified by MALDI-TOF mass spectrometry. Five potential interacting proteins could be identified by using the MASCOT database, YCL058W-A, YLL033W, YOL045W, YKR001C and YJL082W. Beside these proteins a number of non-native proteins were detected, i.e. chaperones and cytosolic enzymes. YCL058W-A is a protein with unknown function. Two variants of this protein exist: YCL058W-A and YCL058C. By modification of this protein with a HA-tag it was possible to co-precipitate these proteins with Vti1p-TAP showing a weak interaction. This interaction was confirmed by a Yeast-2-Hybrid assay. YLL033W codes for a protein with unknown function. By tagging it with HA a weak interaction was detectable by co-precipitation with Vti1p-TAP. This result was confirmed by a Y-2-H assay. The next candidate was YOL045W, a gene which encodes a serine/threonine kinase and is involved in the sugar metabolism. It was not possible to clarify an interaction with Vti1p. Another candidate was YKR001C. It encodes a protein which acts as a dynamin-like GTPase and is involved in cargo transport into the vacuole and cytoskeleton organisation. The interaction could not be verified by a Y-2-H assay. The last interacting candidate was YJL082W. It encodes for a protein with unknown function. It was detected in mitochondria. An interaction was not detectable by a Y-2-H assay.
In a second project N-terminal truncated Vti1p-mutants should be localised by microscopy to investigate a possible effect on intracellular localisation. The mutants were modified with HA-, GFP- and eGFP-tags. Compartments were stained for co-localisation. It could be shown, that a deletion of the complete N-terminus leads to a mislocalisation to the plasmamembrane and an accumulation in punctured structures within the cytosol. The partly deleted mutant and the wild type showed a localisation of tagged Vti1p around the vacuole and into the vacuole. It could be concluded that the N-terminus has a function as a sorting- or recruiting-signal for Vti1p.
The third project was a cooperation between the groups of BCIII and PCIII. It dealt with the efficient expression of the light-inducible cation channel protein Channelrhodopsin-2 in the methylotrophic yeast Pichia pastoris. The protein was modified with a RGS-6HIS-tag for efficient purification by affinity chromatography. The purified protein was destined for functional analysis by IR-spectroscopy. To analyse the function of Channelrhodopsin-2 in more detail, single amino acid mutants were created to study their influence on Channelrhodopsin-2 function.
DA  - 2009
KW  - , Saccharomyces cerevisiae , SNARE Vti1p , N-Terminus , Pichia pastoris , Channelrhodopsin-2 ,
LA  - ger
PY  - 2009
TI  - Identifizierung von Interaktionspartnern und Funktion des N-Terminus des Qb-SNAREs Vti1p in Saccharomyces cerevisiae und Produktion von Channelrhodopsin-2 in Pichia pastoris
UR  - https://nbn-resolving.org/urn:nbn:de:hbz:361-15504
Y2  - 2024-11-22T03:45:03
ER  -