Rab7 and Accessory Protein Function in Endocytosis

University Of New Mexico Health Sciences Center, Albuquerque NM

Investigators

Abstract

This project addresses three specific aims relevant to understanding late endocytic transport and sorting in mechanistic detail. This work is relevant for understanding membrane transport mechanisms and the regulation of receptor trafficking. Studies in Dr. Wandinger-Ness's laboratory have identified Rab7 and its interacting partners (lipid kinases and phosphatases and a proteasome subunit) as a key regulators of late endocytic transport and sorting. They have also demonstrated that late endosomal motility on microtubules is bidirectional and dependent on functional Rab7. Since individual Rab GTPases have largely distinct rather than shared effectors, a comprehensive understanding of membrane trafficking will require the detailed study of individual transport steps and molecules involved. Having identified several key components in late endocytic transport and sorting with support provided by MCB9982161, her plan is to establish in mechanistic terms how these components function together to orchestrate and regulate late endosomal transport and sorting. Planned studies will examine phophoinositide metabolism, proteasome function and microtubule transport all in the context of how these are coordinately regulated by Rab7 and its interacting partners on late endosomes. Based on evidence that the hVPS34 lipid kinase and its p150 adapter form mutually exclusive complexes with the myotubularin 1 lipid phosphatase or with Rab7, Dr. Wandinger-Ness hypothesizes that these selective interactions serve to coordinately regulate local phosphatidyl inositol 3-phosphate (PI3P) levels. In Aim1, she will use in vitro and in vivo assays to test the postulate and establish the mechanisms governing late endosomal phosphoinositide metabolism. Because phosphatidyl inositides play a central role in transport by serving as membrane platforms for recruiting molecules that facilitate membrane fusion and intraluminal vesicle formation, addressing how phosphoinositides are synthesized and turned over will provide new insights into how this aspect of transport is regulated. XAPC7 is an alpha subunit of the proteasome that has been shown by Dr. Wandinger-Ness to complex with Rab7. Based on her initial published data, the proteasome seems to negatively regulate transport, but the exact function of the proteasome on late endosomes remains unclear. Aim2 is geared toward determining the function of the proteasome in the regulation of transport. It is expected that these studies will interface with the growing literature implicating ubiquitination and proteasome activity in the proper trafficking and downregulation of membrane signalling receptors. Finally, data from Dr. Wandinger-Ness' own work and others demonstrate that functional Rab7 and its interacting partner RILP are important for centrosome-directed late endosomal motility on microtubules. Aim3 will explore how the dynactin associated/RILP complex and the KIF1Bb kinesin motor may cooperate to regulate bidirectional late endosomal motility. Together, these studies promise to offer a more comprehensive understanding of late endosomal transport and sorting that is a fundamental facet of the life of eukaryotic cells. In addition, NSF funding of this project will continue to facilitate resource development as exemplified by past contributions in reagents, educational training and resource development. The studies will continue to expand the repertoire of reagents that Dr. Wandinger-Ness has already freely disseminated to over 100 investigators, resulting in numerous citations. The research serves as an underpinning for educational innovation in an interdisciplinary graduate training program. Prior NSF funding provided seed monies for the establishment of a state-of-the-art microscopy facility at the University of New Mexico that is now heavily utilized by students and fellows in various Federally funded research projects. Finally, the project has with prior support been central to the training of a total of 21 undergraduate and graduate students and postdoctoral fellows, more than half of which are women and underrepresented minorities. It is expected this extensive training track record will be expanded with this renewal support.