enzyme precursors in coated vesicles derived from the exocytic and endocytic pathways.

zymes, cathepsin C and cathepsin D, have been examined in lysosomes and coated vesicles (CVs) of rat liver. In addition, the relative proportion of these lysosomal enzymes residing in functionally distinct CV subpopulations was quantitated. CVs contained newly synthesized precursor forms of the enzymes in contrast to lysosomes where only the mature forms were detected. Exocytic and endocytic CV subpopulations were prepared by two completely different protocols. One procedure, a density shift method, uses cholinesterase to alter the density of CVs derived from exocytic or endocytic pathways. The other relies on electrophoretic heterogeneity to accomplish the CV subfractionation. Subpopulations of CVs prepared by either procedure showed similar results, when examined for their relative proportion of cathepsin C and cathepsin D precursors. Within the starting CV preparation, exocytic CVs contained approximately 80-90% of the total steady-state levels of these enzymes while the level in the endocytic population was ~10-13 %. The implications of these findings are discussed with regard to lysosome trafficking. T HE transport of lysosomal enzymes from their site of synthesis in the rough endoplasmic reticulum to the lysosome is a complex, multistep process. During transport, the enzymes pass through numerous cellular compartments and along the way to the lysosome they are modified by the addition of carbohydrate side chains containing mannose 6-phosphate (M6P) j moieties which serve as recognition markers for the binding of the enzymes to specific membrane receptors (M6P receptors). These receptors participate in enzyme sorting and they are thought to play a role in enzyme transport as well. The structure of the recognition markers, and the biosynthetic reactions which lead to their formation, are understood in considerable detail. In addition, much is known about the structure and the physical characteristics of the specific lysosomal enzyme receptors (for reviews see references 18 and 30). Little is known, however, about the details of enzyme sorting and transport. Considerable evidence indicates that clathrin-coated vesicles (CVs) are involved in the process of protein transport (for review see references 7, 8, and 13). Furthermore, early S. Helmy's present address is Faculty of Medicine, Suez Canal University, Ismailia, Egypt. K. yon Figura's present address is Georg-August-Universit~t, G6ttingen, Federal Republic of Germany. 1. Abbreviations used in this paper: CV, clathrin-coated vesicle; M6P, mannose 6-phosphate. morphological studies suggest that these vesicles are also involved in lysosomal enzyme transport (10, 15, 24). CVs isolated from rat liver, calf brain, and human placenta have been shown to contain newly synthesized lysosomal enzymes (5, 29) bound to M6P receptors (4, 28). Localization at the EM level has shown that in rat liver, the coated vesicles containing M6P receptors appear to be concentrated in the trans-Golgi region (11), however, they are also detected in the area of the cis-Golgi (2). Recent studies suggest that coated vesicles shuttle M6P receptors between the Golgi and a discrete population of endosomes (3). Coated vesicles derived from the plasma membrane by receptor-mediated endocytosis also contain M6P receptors (33, 34). Two different procedures have been devised that allow fractionation of rat liver coated vesicles into functionally distinct subpopulations. One procedure uses a cholinesterasemediated density shift technique to alter the density of coated vesicles derived from exocytic or endocytic pathways (14). The other uses agarose gel electrophoresis to separate exocytic and endocytic coated vesicles by their apparent differences in surface charge (17). Because lysosomal enzymes are synthesized at extremely low levels in the cell it would be difficult to use biosynthetic pulse-labeling experiments for detection of newly synthesized lysosomal enzymes in isolated subpopulations of CVs. However, the availability of specific antibodies directed against two rat liver lysosomal © The Rockefeller University Press, 0021-9525/87/06/1743/6 $1.00 The Journal of Cell Biology, Volume 104, June 1987 1743-1748 1743 enzymes has allowed us to examine the relative proportions and the molecular forms of these two lysosomal enzymes in steady-state populations of exocytic and endocytic CVs. Materials and Methods The iodination reagent Iodogen was purchased from Pierce Chemical Co. (Rockford, IL) and carrier free Na]25I was from Amersham Corp. (Arlington Heights, IL). PD-10 columns were from Pharmacia Fine Chemicals (Piscataway, NJ). Immunoprecipitin was from Bethesda Research Laboratories (Bethesda, MD). The Ig fraction of a rabbit antiserum against rat liver cathepsin C (21) was provided by Dr. E Mainferme, University of Namur. The rabbit antiserum and the affinity-purified antibodies against rat liver cathepsin D were provided by Dr. E Baccino, University of Torino. Coated Vesicle and L ysosome Purification Coated vesicles were isolated by a modification (14, 17) of the method of Blitz et al. (1). As previously demonstrated (26), the CVs were at least 95% pure by morphological criteria using both thin section and negative stain EM. Over 95% of the cholinesterase activity was demonstrated to be associated with CVs (14). Coated vesicles were then subjected to the cholinesterase density shift protocol as described (14) or fractionated into subpopulations by preparative agarose gel electrophoresis which purifies CVs to apparent homogeneity (16, 17, 27). Rat liver lysosomes were prepared by applying a light mitochondrial fraction (6) to a discontinuous metrizamide gradient (32). Iodination of Vesicles Purified coated vesicles and lysosomes were solubilized in 50 mM Tris, pH 7.4, 0.15 M NaCI, 4.0 M urea, 1% CHAPS, and labeled for 15 min at room temperature using the Iodogen procedure (22). Free iodide was removed on PD-10 columns equilibrated with 50 mM Tris, pH 7.4, 0.15 M NaC1, 0.5% Nonidet P-40, 0.05 % SDS. Specific activities were typically between 1 and 4 × 106 cpm/Ixg. For iodination of subpopulations of CVs, equal amount of proteins were used. The specific activities of the various subpopulations differed by less than +15%. Figure 1. Molecular forms of cathepsin D in lysosomes and coated vesicles. Lysosomes (Lys) and coated vesicles (CV) isolated from rat liver were solubilized, iodinated, and subjected to immunoprecipitation with cathepsin D antiserum. The precursor (P) and mature (M) forms of rat cathepsin D are indicated. The following 14C-methylated protein standards were used: phosphorylase B, 97,400; bovine serum albumin, 69,000; ovalbumin, 46,000; carbonic anhydrase, 30,000; and cytochrome C, 12,300. Isolation and Analysis of lmmunoprecipitates Iodinated samples were adjusted to 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM iodoacetamide, and 0.5% Triton X-100, and were preabsorbed by incubating at 4°C with 20 mg of Immunoprecipitin overnight and then with 10 mg of Immunoprecipitin for 30 min. The samples were subjected to centrifugation at 45,000 g for 1 h. Cathepsin C was immunoprecipitated from the supernatants as described (21). In some experiments the specificity of the immunoprecipitation was examined by adding an excess of purified rat liver cathepsin D or replacing anti-cathepsin C Ig with preimmune Ig. After immunoprecipitation of cathepsin C, the samples were incubated for 1-4 h with 10 mg Immunoprecipitin and subjected to centrifugation as above. Cathepsin D was immunoprecipitated from the supernarants with either antiserum or afffinity-purified antibodies using the same conditions as for cathepsin C (21). Metabolic labeling of Morris hepatoma 7777 cells with [35S]methionine and immunoprecipitation of cathepsin D from extracts of cells and medium was carried out as described for cathepsin C in reference 21. The cathepsin C immunoprecipitates were solubilized in the absence of dithiothreitol (DTT) and analyzed in 15 % gels, while the cathepsin D immunoprecipitates were solubilized in the presence of 10 mM DTT and analyzed in 12.5% gels by SDS PAGE (19) followed by fluorography (20). Radioactivity in polypeptides was quantified by densitometry of fluorograms.