O-Linked GlcNAc addition and phosphorylation may compete for sites on nuclear pore proteins and transcription factors. We sequenced O-linked GlcNAc transferase from rabbit blood and identified the homologous Caenorhabditis elegans transferase gene on chromosome III. We then isolated C. elegans and human cDNAs encoding the transferase. The enzymes from the two species appear to be highly conserved; both contain multiple tetratricopeptide repeats and nuclear localization sequences. The C. elegans transferase accumulated in the nucleus and in perinuclear aggregates in overexpressing transgenic lines. O-Linked GlcNAc transferase activity was also elevated in HeLa cells transfected with the human cDNA. At least four human transcripts were observed in the tissues examined ranging in size from 4.4 to 9.3 kilobase pairs. The two largest transcripts (7.9 and 9.3 kilobase pairs) were enriched at least 12-fold in the pancreas. Based on its substrate specificity and molecular features, we propose that O-linked GlcNAc transferase is part of a glucose-responsive pathway previously implicated in the pathogenesis of diabetes mellitus.

The dynamins are 100-kDa GTPases involved in the scission event required for formation of endocytotic vesicles. The two main described mammalian dynamins (dynamin؊1 and dynamin؊2) both contain a pleckstrin homology (PH) domain, which has been implicated in dynamin binding to (and activation by) acidic phospholipids, most notably phosphoinositides. We demonstrate that the PH domains of both dynamin isoforms require oligomerization for high affinity phosphoinositide binding. Strong phosphoinositide binding was detected only when the PH domains were dimerized by fusion to glutathione S-transferase, or via a single engineered intermolecular disulfide bond. Phosphoinositide binding specificities agreed reasonably with reported effects of different phospholipids on dynamin GTPase activity. Although they differ in their ability to inhibit rapid endocytosis in adrenal chromaffin cells, the dynamin؊1 and dynamin؊2 PH domains showed identical phosphoinositide binding specificities. Since oligomerization is required for binding of the dynamin PH domain to phosphoinositides, it follows that PH domain-mediated phosphoinositide binding will favor oligomerization of intact dynamin (which has an inherent tendency to self-associate). We propose that the dynamin PH domain thus mediates the observed cooperative binding of dynamin to membranes containing acidic phospholipids and promotes the self-assembly that is critical for both stimulation of its GTPase activity and its ability to achieve membrane scission.

Oculocutaneous albinism type 1TS is caused by mutations that render the melanocyte-specific enzyme tyrosinase temperature-sensitive (ts); the enzyme is inactive in cells grown at 37°C but displays full activity in cells grown at 31°C. To distinguish whether the ts phenotype of the common R402Q variant of human tyrosinase is due to altered enzymatic activity or to misfolding and a defect in intracellular trafficking, we analyzed its localization and processing in transiently transfected HeLa cells. R402Q tyrosinase accumulates in the endoplasmic reticulum (ER) at 37°C but exits the ER and accumulates in endosomal structures in cells grown at 31°C. The inability of the R402Q variant to exit the ER is confirmed by the failure to acquire endoglycosidase H resistance at 37°C and cannot be accounted for solely by enhanced proteasome-mediated degradation. ER retention at 37°C is mediated by the lumenal domain of R402Q tyrosinase, is not dependent on tethering to the membrane, and is irreversible. Finally, a wild-type allelic form of tyrosinase is partially ts in transiently transfected HeLa cells. The data show that human tyrosinase expressed in non-melanogenic cells folds and exits the ER inefficiently and that R402Q tyrosinase exaggerates this defect, resulting in a failure to exit the ER at physiologic temperatures.

Distinct cytoplasmic sorting signals target integral membrane proteins to late endosomal compartments, but it is not known whether different signals direct targeting by different pathways. The availability of multiple pathways may permit some cell types to divert proteins to specialized compartments, such as the melanosome of pigmented cells. To address this issue, we characterized sorting determinants of tyrosinase, a tissue-specific resident protein of the melanosome. The cytoplasmic domain of tyrosinase was both necessary and sufficient for internalization and steady state localization to late endosomes and lysosomes in HeLa cells. Mutagenesis of two leucine residues within a conventional di-leucine motif ablated late endosomal localization. However, the properties of this di-leucine-based signal were distinguished from that of CD3␥ by overexpression studies; overexpression of the tyrosinase signal, but not the well characterized CD3␥ signal, induced a 4-fold enlargement of late endosomes and lysosomes and interfered with endosomal sorting mediated by both tyrosine-and other di-leucine-based signals. These properties suggest that the tyrosinase and CD3␥ dileucine signals are distinctly recognized and sorted by distinct pathways to late endosomes in non-pigmented cells. We speculate that melanocytic cells utilize the second pathway to divert proteins to the melanosome.