We have used the pH-dependent fluorochrome fluorescein-dextran (FD) to study the acidification of prelysosomal vacuoles (endosomes) and lysosomes isolated from cultured macrophages and fibroblasts. FD was internalized by pinocytosis under conditions that allowed its selective localization in endosomes (1-to 5-min pulse) or in lysosomes (5-min pulse, 30-min chase). Fibroblasts were also exposed to FD at 20°C, at which temperature endosome-lysosome fusion is inhibited. Cells were homogenized and labeled organelles were separated by centrifugation in Percoll density gradients. The addition of ATP rapidly decreased the internal pH of both endosomes and lysosomes, as indicated by a decrease in fluorescence intensity. The pH gradient was dissipated by H+ ionophores and ammonium.chloride. Acidification was not affected by inhibitors of the mitochondrial F1,F0-ATPase or the Na+, K, K+-ATPase and did not require permeant anions, Na+, or KV. Of the inhibitors tested, only N-ethylmaleimide prevented the ATP-dependent acidification of both compartments. These findings provide direct support for the existence of an acidic prelysosomal compartment that may be acidified via the same type of H+ pump believed to operate in lysosomes and.secretory granules.Pinocytosis typically results in the accumulation and degradation of internalized macromolecules in secondary lysosomes (1). However, delivery to lysosomes requires the participation of at least two other classes of endocytic vacuoles. Pinocytic vesicles, often with clathrin-containing coats, form at the plasma membrane and constitute the primary endocytic compartment. These vesicles subsequently fuse with, or fuse together to form, a somewhat larger class of secondary endocytic vacuoles (0.2-1.0 ,um in diameter), referred to here as endosomes (1,2). Endosomes can be distinguished from lysosomes in several ways: (i) they have a relatively low buoyant density (3-8); (ii) they are labeled by pinocytic markers more rapidly than and prior to labeling of lysosomes (7,9); (iii) internalized macromolecules exhibit only a transient residence in endosomes, whereas lysosomes are typically the final destination (9); and (iv) endosomes are relatively devoid of acid hydrolases (3,(6)(7)(8).These differences notwithstanding, endosomes may be similar to secondary lysosomes in at least one significant characteristic, low internal pH. This has been suggested by recent studies in which receptor-bound ligands were coupled to fluorescein (whose fluorescence spectrum is a titratable function of pH) and found to be internalized into an acidic compartment prior to delivery to lysosomes (8,10). This was the case also with Semliki Forest virus (SFV), which requires a pH of <6 for fusion and penetrates into the cytosol from prelysosomal vacuoles (11). Here, we have used isolated endosomes labeled with the pH-sensitive fluorochrome fluorescein-dextran (FD) (12, 13) to study more directly the acidification of prelysosomal vacuoles. Two points are made: (i) both isolated endosomes and lysosomes c...

A Chinese hamster ovary cell mutant DTG 1-5-4, was selected for pleiotropic defects in receptor-mediated endocytosis by methods previously described (Robbins, A. R., S. S. Peng, and J. L. Marshall, 1983, J. Cell Biol., 96:1064-1071. DTG 1-5-4 exhibited increased resistance to modeccin, Pseudomonas toxin, diphtheria toxin, Sindbis virus, and vesicular stomatitis virus, as well as decreased uptake via the mannose 6-phosphate receptor. Fluorescein-dextran-labeled endosomes isolated from DTG 1-5-4 were deficient in ATP-dependent acidification in vitro. Endocytosis and endosome acidification were both restored in revertants of DTG 1-5-4 and in hybrids of DTG 1-5-4 with DTF 1-5-1, another endocytosis mutant exhibiting decreased ATP-dependent endosome acidification.Both DTG 1-5-4 and DTF 1-5-1 were blocked at two stages of infection with Sindbis virus: at low multiplicities of infecting virus, resistance reflected a block in viral penetration into the cytoplasm, but at higher multiplicities of infection the block was in virus release. Like endocytosis, release of Sindbis virus was increased in revertants of DTG 1-5-4 and in DTG 1-5-4 x DTF 1-5-1 hybrids. Decreased release of virus from DTG 1-5-4 correlated with defects in some of the Golgi apparatus-associated steps of Sindbis glycoprotein maturation: proteolytic processing of the precursor pE2, galactosylation, and transport to the cell surface all were inhibited. In contrast, mannosylation, fucosylation, and acylation of the Sindbis glycoproteins, and galactosylation of vesicular stomatitis virus and cellular glycoproteins occurred to similar respective extents in mutant and parent. Electron microscopic examination of Sindbis-infected DTG 1-5-4 showed a remarkable accumulation of nucleocapsids bound to cisternae adjacent to the Golgi apparatus; virions were observed in the lumina of some of these cisternae.That the alterations in both endocytosis and Golgi-associated steps of viral maturation result from a single genetic lesion indicates that these processes are dependent on a common biochemical mechanism. We suggest that endocytic and secretory pathways may share a common component involved in ion transport.

At least four distinct forms of clathrin light chains are found in mammalian cells. This molecular variability derives from tissue-specific patterns of expression of LCa and LCb genes. Sequence analysis shows an overall homology of 60% between LCa and LCb and the presence of brain-specific insertion sequences. These findings suggest that the different light chains have both shared and specialized functions. To address this question we have used a panel of monoclonal antibodies to identify two structurally and functionally distinct regions in the clathrin light-chain sequences. One region (residues 158-208) is exposed in native clathrin structures (triskelions and coated vesicles) and includes the brain-specific insertion sequences. The second region (residues 93-157), which is cryptic in native clathrin structures, is involved in binding the clathrin heavy chain and contains the region of strongest homology with intermediate filament proteins.

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