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Dissecting the roles of O-glycosylation and dimerization
in the apical sorting of a model raft-independent protein
Robert Youker 1 , Federick Lanni 2, 3 , Haibing Teng 3 , Ora Weisz 1 .
Renal Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh PA
15261 1 , Department of Biological Sciences, Carnegie Mellon University, Pittsburgh PA 15213 2 ,
Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh PA 15213 3 ,
Abstract:
Proteins sorted to the apical plasma membrane in polarized epithelial cells can be categorized
into two classes based on their differential association with cholesterol and glycolipid-enriched
membrane microdomains commonly termed “lipid rafts”. Our laboratory and others have
previously demonstrated that lipid raft-associated and lipid raft-independent proteins take
different routes to the apical surface (1, 2). Interestingly, raft-associated and raft-independent
apical cargoes appear to be segregated into distinct subdomains even within the trans-Golgi
network (TGN) (3). Moreover, TGN export requirements of the raft-independent protein p75
assessed using an in vitro assay are markedly different from that of a raft-associated protein.
Self-association or “clustering” is important for the sorting of raft-associated proteins but the
role of clustering in raft-independent sorting is unclear. The apical sorting determinant in p75
resides within its O-glycosylated stalk, and galectin-mediated crosslinking has been proposed as
a sorting mechanism for this protein (4). Additionally, a novel dimerization motif (Cys257,
A261XXXG266) has been identified in the transmembrane region of p75 that could contribute to
the formation of oligomers (5). I have created a panel of p75 mutants to determine the roles of
O-glycosylation and dimerization in the segregation and export characteristics of p75 staged in
the TGN. In vitro TGN budding assays and immunofluorescence microscopy are being employed
to examine segregation and TGN export of mutants compared with wild type p75. In addition
the local cellular environment occupied by wild type and mutant proteins is being explored
using biophysical approaches. 1) Cresawn, K. O. et al. (2007) EMBO J. 26, 3737-3748 2) Jacob,
R., and Naim, H. Y. (2001) Curr. Biol. 11, 1444-1450 3) Guerriero, C. J. et al. (2008) JBC 283,
18040-7 4) Delacour et al. (2007) Traffic 8, 379-388 5) Vilar, M. et al. (2009) Neuron 62, 72-83.