The Golgi localization of the carbohydrate sulfotransferases.

摘要

Sulfation of endothelial glycoproteins is a regulatory modification that promotes the binding of the leukocyte adhesion molecule L-selectin. Certain members of a family of related sulfotransferases, known as the GlcNAc6STs, have been implicated in the biosynthesis of L-selectin ligands. When tested in vitro, the enzymes act on similar carbohydrate substrates, but they have been shown to function on discrete glycoproteins in vivo . I hypothesized that the specificity of the sulfotransferases was due to their confinement in different compartments of the Golgi apparatus, an organelle that houses most of the enzymes responsible for the posttranslational modifications found on secreted and cell surface proteins.;In Chapter 2 I demonstrate that GlcNAc6ST-1, -2 and -3 have distinct Golgi localizations, with GlcNAc6ST-1 confined to the trans-Golgi network, GlcNAc6ST-3 confined to the early secretory pathway and GlcNAc6ST-2 distributed throughout the Golgi. Their localization was correlated with preferred activity on either N-linked or O-linked glycoproteins. I propose a model in which Golgi enzyme localization and competition orchestrate the biosynthesis of L-selectin ligands.;In Chapter 3 I probe the importance of the stem region of GlcNAc6ST-1 with respect to substrate preference, localization, and oligomerization. Analysis of truncation mutants demonstrated that perturbation of the stem region of GlcNAc6ST-1 affects the cellular substrate preference of the enzyme without altering Golgi localization. Two cysteine residues within the stem and transmembrane domains were found to be critical for dimerization of the enzyme. Collectively, these results indicate that the stem region of GlcNAc6ST-1 directly affects substrate specificity, independent of dimerization or Golgi localization.;In chapter 4 I describe a system for conditional activation of Golgi-resident sulfotransferases using a chemical inducer of dimerization. The approach capitalizes on two features shared by a majority of these enzymes: their requirement of Golgi localization for activity on cellular substrates and the modularity of their catalytic and localization domains. Fusion of these domains to the proteins FRB and FKBP enabled their induced assembly by the natural product rapamycin. This approach provides a new means for studying sulfate-dependent processes in cellular systems and, potentially, in vivo.