Date of Award

Spring 2009

Document Type

Restricted Thesis

Terms of Use

© 2009 Caitlin Mullarkey. All rights reserved. Access to this work is restricted to users within the Swarthmore College network and may only be used for non-commercial, educational, and research purposes. Sharing with users outside of the Swarthmore College network is expressly prohibited. For all other uses, including reproduction and distribution, please contact the copyright holder.

Degree Name

Bachelor of Arts

Department

Biology Department

Abstract

NMB is a putative melanosomal integral membrane protein that is expressed in a diverse range of cell types including retinal pigment epithelium, osteoblasts, and macrophages. The GPNMB gene was identified fourteen years ago, but characterization of the NMB gene product remains lacking. Despite being linked to multiple pathologies including pigmentary glaucoma and glial tumors, the function, subcellular location, and sorting of NMB have yet to be fully described. Here we show basic biochemical and cytological characterization of NMB in an endogenous retinal pigment epithelial system. To elucidate the trafficking and subcellular localization of the protein, we transfected pigment and non-pigment cells with NMB constructs and analyzed transfectants by indirect immunofluorescence microscopy. Our data demonstrate that in ARPE-19 pigment cells NMB is segregated away from lysosomes at steady state. Expression of NMB in non-pigmented COS-7 cells reveals partial overlap with LAMP-1 positive structures indicating co-localization with lysosomes. The NMB cytoplasmic domain contains within it a dileucine-based motif that is known to function as endocytic signal in other melanosomal proteins. Localization and continuous antibody uptake experiments with a site-directed mutant show that loss of the dileucine-based signal leads to an endocytic defect resulting in the accumulation of NMB on the cell surface. From these data we propose that NMB is sorted via the cell surface to its intracellular site of residence.

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