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The limiting membrane of the lysosome contains a group of transmembrane glycoproteins named lysosome-associated membrane proteins (Lamps). These proteins are targeted to lysosomes by virtue of tyrosine-based sorting signals in their cytosolic tails. Four adaptor protein (AP) complexes, AP-1, AP-2, AP-3, and AP-4, interact with such signals and are therefore candidates for mediating sorting of the Lamps to lysosomes. However, the role of these complexes and of the coat protein, clathrin, in sorting of the Lamps in vivo has either not been addressed or remains controversial. We have used RNA interference to show that AP-2 and clathrin-and to a lesser extent the other AP complexes-are required for efficient delivery of the Lamps to lysosomes. Because AP-2 is exclusively associated with plasma membrane clathrin coats, our observations imply that a significant population of Lamps traffic via the plasma membrane en route to lysosomes.

The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at presynaptic terminals but is also present in cell bodies. unc-11 mutants are defective in two aspects of synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to synaptic vesicles. The reduction of synaptobrevin at synaptic vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second, unc-11 mutants accumulate large vesicles at synapses. We propose that the UNC-11 protein mediates two functions during synaptic vesicle biogenesis: it recruits synaptobrevin to synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly.

The light chain subunits of clathrin, LCa and LCb, have been implicated in the regulation of coated vesicle disassembly and other aspects of clathrin cycling within the cell. The potential for functional specialization of each light chain is suggested by tissue-specific variation in the relative amounts of the two light chains and by conservation of differences between LCa and LCb sequences during evolution. To investigate whether there might be exclusive roles for LCa and LCb in clathrin function, the expression of LCa was manipulated in C1R lymphoid cells and PC12 pheochromocytoma cells by transfection with light chain cDNA. These two cell lines differ in their ratios of LCa to LCb, expressing 86 and 25% LCa, respectively. After transfection with exogenous human LCa cDNA, a PC12 cell derivative was produced that completely lost the ability to manufacture LCa. Loss of LCa expression was found to be because of gene disruption and consequent lack of mRNA transcription. In C1R cells, the normally high level of LCa expression was reduced to 25% by overexpression of transfected LCb cDNA under the control of an inducible promoter. The C1R transfectants with reduced levels of LCa and the LCa-negative PC12 transfectant grow normally and show no change in clathrin distribution, clathrin assembly level, or impairment of endocytosis or secretion compared with wild-type cells and cells transfected with vectors lacking light chain cDNA. However, subtle alterations in the hsc70-mediated clathrin uncoating process were observed for vesicles derived from the LCa-negative cells, reflecting the preferential activity of LCa in stimulating the in vitro uncoating reaction.