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UBC Theses and Dissertations

Using large-scale screens to identify novel regulators of endosomal trafficking in yeast Dalton, Lauren


Cellular protein trafficking, the concerted action of moving proteins to the appropriate cellular location is important for the proper functioning of the cell. Proteins are sorted at hubs such as the endosome, where they are targeted either for degradation or for recycling to the Golgi. Yeast is an excellent model organism to study protein trafficking due to the conservation with higher eukaryotes and the ease of genetic manipulation. In this thesis, two different high-throughput approaches were used to study the protein machinery that controls the yeast endosomal sorting network. First, correlation analysis, which compares genome-wide phenotypic profiles, was used to uncover new aspects of endosomal sorting and two specific examples were chosen for further study. In the first example, we explored positive and negative genetic interaction profile correlations for members of the uncharacterized yeast complex, BLOC, which suggested a role in endosomal sorting. In particular, we found that BLOC was needed for proper downregulation of a manganese transporter similar to ESCRT (Endosomal Sorting Complex Required for Transport, a well-studied endosomal sorting complex. In the second example, we explored the predictive value of negatively correlated genetic interaction profiles for gene mutants within known protein complexes and found that these negative correlations described two types of regulatory interactions between the resulting proteins, direct inhibition and competition for shared subunits. We then showed that the previously uncharacterized VID Associated Factor 1 (Vaf1) was negatively correlated with respect to its genetic interactions and downregulated by the VID complex. In summary, correlation analysis provides a robust tool to identify the functional relationship between proteins. Second, a quantitative genome-wide endosomal sorting screen followed by a secondary high-throughput microscopy screen, uncovered novel endosomal regulators of the flippase, Neo1. Loss of these regulators caused three phenotypic outcomes: reduced recycling from endosomes, delayed endosomal progression, or reduced colocalization with known binding partners. In particular, a short motif in the N-terminus of Neo1 was found to be necessary for Snx3 dependent sorting, correct sorting of other Snx3 cargos, and full Neo1 function. Overall, this study illuminates the power of high-throughput screens to discover new regulators of endosomal sorting.

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