Characterizing Adaptor Protein Complex 3 and Its Interactions with Cellular Proteins

Nack, Ayla L

Undergraduate Honors Thesis

Characterizing Adaptor Protein Complex 3 and Its Interactions with Cellular Proteins Público Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/qv33rz38p

Abstract

Adaptor Protein Complex 3 (AP-3) is a heterotetrameric protein complex involved in the transport of proteins from the Golgi to the lysosome. AP-3 is known to interact with other cellular proteins during its activity, and other interactors are anticipated. This study investigates additional candidate AP-3 interactors in the budding yeast Saccharomyces cerevisiae that were identified by three screening methods: mass spectrometry of proteins in yeast cell extracts that copurify with recombinant AP-3 proteins, genetic screening for mutations in yeast that missort an AP-3 reporter cargo protein, and in-vivo proximity-labeling of yeast cellular proteins by AP-3 subunits fused to biotin ligase. The candidate AP-3 interactors are evaluated using bimolecular fluorescence complementation (BiFC), which revealed 17 positive AP-3 interactors out of 102 potential candidates. One of these interactors is Vma10, a subunit of the vacuolar ATPase (V-ATPase). The interaction between AP-3 and the V-ATPase is investigated through location-specific V-ATPase subunits Vph1 and Stv1. BiFC revealed that Vph1 is a strong AP-3 interactor, whereas Stv1 is not. Using confocal microscopy, we show that Vph1 is transported to the vacuole (the yeast lysosome) via the VPS Pathway, which is an alternative transport pathway that parallels the route of delivery via the AP-3 pathway. Additionally, this study shows that expression of the Apl5 subunit of AP-3 in vivo requires expression of each of the other 3 AP-3 subunits, suggesting that Apl5 might be the last subunit to coassemble during AP-3 complex formation.

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