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.

Surfactant protein B (SP-B8), an 8-kDa hydrophobic protein essential for surfactant and normal lung function, is produced from the intracellular processing of preproSP-B. To characterize SP-B processing in human type 2 cells, we used human fetal lung in explant culture and polyclonal antibodies to human SP-B8(Phe201–Met279) and to specific epitopes within the NH2- and COOH-terminal propeptide domains (Ser145–Leu160, Gln186–Gln200, and Gly284–Ser304). Western blot analysis revealed a novel intermediate at ∼9 kDa, representing mature SP-B8, with a residual NH2-terminal peptide of ∼10 amino acids. Pulse-chase studies showed a precursor-product relationship between the 9- and 8-kDa forms. During differentiation of type 2 cells in explant culture, the rate of proSP-B conversion to 25-kDa intermediate remained constant, whereas the rate of 25-kDa intermediate conversion to SP-B8increased, resulting in a net increase in tissue SP-B8. Dexamethasone did not affect the rate of proSP-B processing but markedly enhanced the rate of SP-B8 accumulation. We conclude that NH2-terminal propeptide cleavage of proSP-B is a multistep process and that more distal processing events are rate limiting and both developmentally and hormonally regulated.

tGolgin-1 (golgin-245, trans golgi p230) and golgin-97 are members of a family of peripheral membrane proteins of unknown function that localize to the trans Golgi network (TGN) through a conserved C-terminal GRIP domain. We have probed for GRIP protein function by assessing the consequences of overexpressing isolated GRIP domains. By semi-quantitative immunofluorescence microscopy we found that high level expression of epitope-tagged, GRIP domain-containing fragments of tGolgin-1 or golgin-97 specifically altered the characteristic pericentriolar distribution of TGN integral membrane and coat components. Concomitantly, vesicular transport from the TGN to the plasma membrane and furin-dependent cleavage of substrate proteins in the TGN were inhibited. Mutagenesis of a conserved tyrosine in the tGolgin-1 GRIP domain abolished these effects. GRIP domain overexpression had little effect on the distribution of most Golgi stack resident proteins and no effect on markers of other organelles. Electron microscopy analyses of GRIP domain-overexpressing cells revealed distended perinuclear vacuoles and a proliferation of multivesicular late endosomes to which the TGN resident protein TGN46 was largely mislocalized. These studies, the first to address the function of GRIP domain-containing proteins in higher eukaryotes, suggest that some or all of these proteins and/or their ligands function in maintaining the integrity of the TGN by regulating resident protein localization.