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A specialized exit route: specific recognition and cell cycle-dependent transport of exomer-dependent cargoes in "Saccharomyces cerevisiae"

Rockenbauch, Uli. A specialized exit route: specific recognition and cell cycle-dependent transport of exomer-dependent cargoes in "Saccharomyces cerevisiae". 2012, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_9933

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Abstract

In yeast, the exomer complex – consisting of Chs5p and the ChAPs family – mediates the exit of a subset of cargoes from the trans-Golgi network via secretory vesicles. The best-characterized protein of this pathway is Chs3p, which localizes to the bud neck in a cell cycle-dependent manner. This work aimed to create a better understanding of the role of exomer in cargo export to the plasma membrane.
In a candidate-based screen, we found that most of the factors required for correct delivery of Chs3p to the plasma membrane also affected exomer-independent cargoes such as Hxt2p or Pma1p. However, a SRO7 deletion specifically impaired exocytosis of Chs3p-containing vesicles. Moreover, in Deltasro7 as well as Deltaypt31, bud neck localization of Chs3p was particularly affected in large- or small-budded cells, respectively. These findings suggest that traffic to, as well as endocytosis from the plasma membrane, is regulated differently according to the cell cycle stage.
In an attempt to gain a better understanding of how exomer components associate with each other and recognize cargoes, we found that the ChAPs contain five essential tetratricopeptide repeats (TPRs). These repeats are interchangeable between the ChAPs and form a conserved structural backbone. TPR1�4 forms a binding site for Chs5p, while TPR5 may have a more structural role in exomer complex assembly. Surprisingly, using domain-switch chimeras, we found that the cargo specificity of the ChAPs is not determined by a single domain, but rather by large stretches distributed throughout their sequence. Unlike COPI, COPII or clathrin/AP coats, exomer therefore appears to employ an extensive, three-dimensional surface for cargo recognition.
These findings opened up the possibility that exomer-dependent cargoes may be sorted into different secretory vesicles than exomer-independent cargoes. We therefore sought to establish a protocol for differential vesicle immunoprecipitation, which would also allow us to characterize the cargo content of individual vesicle populations. Unfortunately, contaminating endosomes in the vesicle preparation rendered our experiments inconclusive. In parallel, we tested mass spectrometry on purified plasma membranes as an alternative strategy to identifying novel exomer-dependent cargoes. However, this approach turned out to be not ideally suited, as judged by the amount of time and resources required for optimization. We might therefore have to turn to new technologies – or define more suitable methods – in order to be able to characterize vesicle sub-populations.
Advisors:Spang, Anne
Committee Members:Robinson, Margaret
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Growth & Development > Biochemistry (Spang)
Item Type:Thesis
Thesis no:9933
Bibsysno:Link to catalogue
Number of Pages:158 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:49
Deposited On:23 Jul 2012 12:25

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