Research

Receptor Trafficking

Diagram
Mup1-pHluorin is a model plasma membrane receptor that is trafficked to the yeast vacuole/lysosome.
Stained cells
Mup1 is sorted to the lysosome/vacuole lumen; pHluorin signal is quenched
 

We utilize both budding yeast and mammalian cellular systems to study receptor trafficking, and more generally membrane reshaping. As a post-doc in the laboratory of Scott Emr, Ph.D., at Cornell University, Mike conducted a global screen in budding yeast to uncover novel proteins involved in the endolysosomal trafficking of receptors.

We used Mup1-pHluorin, a plasma membrane methionine permease, as a well-characterized model receptor that displays inducible endolysosomal sorting upon addition of exogenous methionine.

Using Mup1-pHluorin, we uncovered genes whose deletion causes the mis-sorting of Mup1-pHluorin to the acidic interior of the yeast lysosome/vacuole.

Future studies will dissect the mechanistic roles of these genes in endolysosomal trafficking, and their roles in mammalian endolysosomal trafficking.

Work Flow of Global Screening for Novel Endolysosomal Sorting Proteins

Diagram
1. Generate Mup1-pHluorin S. cerevisiae yeast KO collection
Diagram
2. Use high-throughput flow cytometry to screen for Mup1-pH sorting defects, using methionine to induce endocytosis
Diagram
3. Visually characterize hits by light microscopy:
Top: Class 1 endocytosis defects
Middle: Class 2 endosomal sorting defects
Bottom: Class 3 MVB biogenesis defects