UBC Theses and Dissertations
Analysis of two members of the EXT family, EXT1 and EXT2 bearing ER-, Golgi-, and secretory vesicle-sorting motifs Lee, Tracy Phek Hui
EXT1 and EXT2 are type II transmembrane glycosyltransferases involved in the biosynthesis of heparan sulfate (HS) polysaccharide chains (McCormick et al., 2000). These two glycoproteins are found predominantly in the ER when overexpressed individually but form a Golgi-localized heterocomplex when co-transfected together (Kobayashi et al., 2000; McCormick et al., 2000). To examine the roles of the N-terminal domains of EXT1 and EXT2, a series of chimeric cDNAs were constructed with ER-, Golgi- and secretory vesicle-sorting motifs. These constructs were transiently expressed in mammalian cells and tested for in vivo HS biosynthetic activity, heterocomplex formation, and subcellular localization. All EXT1 chimeras were able to synthesize HS in vivo as determined by their ability to restore herpes simplex virus infectivity to normally resistant HS-deficient Sog9 cells, though with varying efficiencies. Sec-EXT1 and Sec-EXT2 were able to form a heterocomplex together despite the loss of their wild-type (wt) cytoplasmic N-termini, putative transmembrane domains and 10 aa of their stem regions, suggesting that the N-termini of EXT1 and EXT2 are not essential for heterocomplex formation. The steady state subcellular distribution of the chimeras was determined by indirect immunofluorescence microscopy. As specified by their sorting motifs, ER-EXT1 and ER-EXT2 distributed in staining patterns distinctive of the ER, and Sec-EXT1 and Sec-EXT2 displayed staining patterns indicative of secretory vesicles. Though Golgi-EXT1 and Golgi-EXT2 did result in perinuclear distribution, a majority of transfected cells exhibited staining patterns typically observed for ERGIC- or early Golgi-localization. Interestingly, when each of the EXT1 chimeric constructs was co-transfected with wt EXT2, both proteins co-localized together and exhibited a staining pattern characteristic of the Golgi regardless of their respective sorting signals. This suggests that when a wt anchoring partner is present, chimeric EXT Is are able to form a heterocomplex with wt EXT2 and localize to the Golgi. Taken together, the results presented in this thesis suggest that the major Golgi-localization mechanism used by EXT1 and EXT2 is hetero-oligomer formation as described in the kin recognition model (Nilsson et al., 1993) which hypothesizes that Golgi-resident proteins form complexes too large to enter transport vesicles and are thus retained in the Golgi.
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