Amutation in the small GTPase Rab38 gives rise to the mouse coat color phenotype “chocolate” (cht), implicating Rab38 in the regulation of melanogenesis. However, its role remains poorly characterized. We report that cht Rab38G19V is inactive and that the nearly normal pigmentation in cht melanocytes results from functional compensation by the closely related Rab32. In cht cells treated with Rab32-specific small interfering RNA, a dramatic loss of pigmentation is observed. In addition to mature melanosomes, Rab38 and Rab32 localize to perinuclear vesicles carrying tyrosinase and tyrosinase-related protein 1, consistent with a role in the intracellular sorting of these proteins. In Rab38/Rab32-deficient cells, tyrosinase appears to be mistargeted and degraded after exit from the trans-Golgi network (TGN). This suggests that Rab38 and Rab32 regulate a critical step in the trafficking of melanogenic enzymes, in particular, tyrosinase, from the TGN to melanosomes. This work identifies a key role for the Rab38/Rab32 subfamily of Rab proteins in the biogenesis of melanosomes and potentially other lysosome-related organelles.

To prevent excessive degradation of internalized antigens, which could destroy the peptides recognized by T lymphocytes, dendritic cells have developed several strategies that limit proteolytic activity in phagosomes. The recruitment of the NADPH oxidase NOX2 prevents acidification of phagosomes, limiting antigen degradation. Here, we show that dendritic cells derived from Rab27a-deficient ashen mice show increased phagosome acidification and antigen degradation, causing a defect in antigen cross-presentation. Enhanced acidification results from a delay in the recruitment to phagosomes of a subset of lysosome-related organelles containing the membrane subunits of NOX2. The Rab27a-dependent recruitment of these "inhibitory lysosome-related organelles" to phagosomes continuously limits acidification and degradation of ingested particles in dendritic cells, thus promoting antigen cross-presentation.

An insulin granule membrane protein-tyrosine phosphatase (PTP) homologue, phogrin, was cloned by expression screening of a rat insulinoma cDNA library. The 3723-base pair cDNA encoded a transmembrane glycoprotein of 1004 amino acids (M r 111876) that underwent post-translational proteolysis to 60 -64-kDa products after a 30-min delay. The kinetics of proteolytic conversion (t1 ⁄2 ‫؍‬ 45 min) and turnover (t1 ⁄2 ‫؍‬ 12 h) were consistent with sorting and conversion in a late compartment of the secretory pathway. Studies on the native ␤-cell protein suggested that the COOH-terminal PTP domain was on the cytosolic face of the secretory granule. The lumenal segment was comprised of a protease-resistant globular domain of around 25 kDa. Its localization and topology is thus consistent with a transmembrane receptor function related to granule biogenesis, exocytosis, or subsequent membrane recovery, and it should prove to be a useful cell biological marker for the granule membrane. High expression of the mRNA (5.4 kilobases) and protein was evident in islets, pancreatic ␣-and ␤-cell tumor lines, brain cells, and other cells of neuroendocrine lineage. It is closely related to the diabetic autoantigen ICA512 (IA-2) (42% identity overall; 80% in the 260-amino acid PTP domain) and thus a potential target of autoimmunity in diabetes mellitus.Integral membrane proteins of intracellular storage vesicles in the regulated pathway of secretion are thought to play a central role in the mechanism of exocytosis and its regulation. They are also presumed to be involved in membrane recycling following exocytosis and in transmembrane signal transduction during the biogenesis, maturation, and senescence of these organelles. The synaptic vesicle has been well characterized, but our knowledge of the membrane proteins of the dense core secretory granule is sketchy and largely confined to proteins that have a tissue-specific function and whose activities relate to maintenance of the lumenal environment. Such molecules include dopamine ␤-hydroxylase and cytochrome b 561 of the chromaffin granule (1), glycoproteins 1 and 2 of pancreatic acinar granules (2), P-selectin in platelets and endothelium (3), and the peptide amidating monoxygenase of cells of a neuroendocrine lineage (4). A new series of dense core granule candidate proteins has recently emerged either as a result of polymerase chain reaction-based searches for homologues of proteins implicated in intracellular membrane traffic or by examining the subcellular distribution of synaptic vesicle membrane proteins in granulated cell types. Small GTP-binding proteins, in particular isoforms of Rab3 (5), as well as the synaptic vesicle constituents synaptotagmin (6) and vesicle-associated membrane protein/synaptobrevin-2 (7, 8) have been documented by such approaches. The need remains, however, to identify other functionally important dense core granule proteins and to establish granule-specific markers for cell biological studies of granule membrane traffic.An alternative approach to the ...

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