We are using Saccharomyces cerevisiae as a model system for understanding how the lysosome is formed and how it mediates the degradation of membrane proteins. One trafficking pathway that contributes to the biogenesis of lysosomes and lysosome-related organelles is controlled by the Adaptor Protein-3 (AP-3) vesicle coat protein complex. In humans, loss of functional AP-3 causes defects in melanogenesis and platelet function and mice without AP-3 also show neurological defects. In yeast, the AP-3 pathway transports a subset of proteins from the Golgi apparatus to the vacuole (lysosome) and allows them to bypass transit through prevacuolar/endosomal compartments. We have conducted a genetic screen to identify other proteins that contribute to the AP-3 trafficking pathway and will now analyze the functional role of these proteins.Proteins that do travel through the prevacuolar/endosomal compartment are subject to a different sorting event. Here they can be incorporated into lumenal membranes of the endosome that are in turn delivered and degraded in the interior of the vacuole. This mechanism ensures the destruction of both cytosolic and lumenal domains of membrane proteins. Not all proteins that travel through the endosome are targeted to lumenal vesicles for degradation. We have found that when Ubiquitin is attached to membrane proteins, it acts as a specific sorting signal for incorporation into endosomal lumenal vesicles. We have also identified a putative sorting receptor for Ubiquitin, which would recognize proteins fated for degradation. This receptor complex also plays a role in sorting proteins that recycle out of endosomes back to the Golgi apparatus. We will now characterize the function of this candidate receptor at the molecular level to define how it carries out its distinct and multiple functions. This analysis will be buttressed by reconstituting the process of lumenal vesicle formation and protein sorting in vitro.
| Krishnamani, Venkatramanan; Peterson, Tabitha A; Piper, Robert C et al. (2018) Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens. J Vis Exp : |
| Peterson, Tabitha A; Stamnes, Mark A; Piper, Robert C (2018) A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions. J Vis Exp : |
| Xu, Peng; Hankins, Hannah M; MacDonald, Chris et al. (2017) COPI mediates recycling of an exocytic SNARE by recognition of a ubiquitin sorting signal. Elife 6: |
| MacDonald, Chris; Piper, Robert C (2017) Genetic dissection of early endosomal recycling highlights a TORC1-independent role for Rag GTPases. J Cell Biol 216:3275-3290 |
| MacDonald, Chris; Winistorfer, Stanley; Pope, Robert M et al. (2017) Enzyme reversal to explore the function of yeast E3 ubiquitin-ligases. Traffic 18:465-484 |
| MacDonald, Chris; Piper, Robert C (2016) Cell surface recycling in yeast: mechanisms and machineries. Biochem Soc Trans 44:474-8 |
| Pashkova, Natasha; Peterson, Tabitha A; Krishnamani, Venkatramanan et al. (2016) DEEPN as an Approach for Batch Processing of Yeast 2-Hybrid Interactions. Cell Rep 17:303-315 |
| MacDonald, Chris; Stamnes, Mark A; Katzmann, David J et al. (2015) Tetraspan cargo adaptors usher GPI-anchored proteins into multivesicular bodies. Cell Cycle 14:3673-8 |
| MacDonald, Chris; Payne, Johanna A; Aboian, Mariam et al. (2015) A family of tetraspans organizes cargo for sorting into multivesicular bodies. Dev Cell 33:328-42 |
| Peterson, Tabitha A; Yu, Liping; Piper, Robert C (2015) Backbone and side-chain NMR assignments for the C-terminal domain of mammalian Vps28. Biomol NMR Assign 9:21-4 |
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