Cycling of two endogenous lysosomal membrane proteins, lamp-2 and acid phosphatase, between the cell surface and lysosomes in cultured rat hepatocytes.
Our previous studies provided evidence that a 107-kDa major lysosomal membrane glycoprotein termed lamp-1 shuttles between lysosomes and the plasma membrane along the endocytic pathway in rat hepatic cells [Furuno et al. (1989) J. Biochem. 106, 708-716; Furuno et al. (1989) J. Biochem. 106, 717-722]. In the present study, we investigated the movement of a 96-kDa major lysosomal membrane glycoprotein, referred to as lamp-2, and lysosomal acid phosphatase (LAP) in the endocytic membrane transport system of cultured rat hepatocytes. Fab' fragments of anti-lamp-2 and anti-LAP antibodies conjugated with horseradish peroxidase (HRP) were used as probes to analyze quantitatively the transport of these two membrane proteins from the cell surface to lysosomes. After the addition of HRP-anti-lamp-2 and anti-LAP Fab' fragments to the culture medium, the delivery of the antibody conjugates to lysosomes was examined by cell fractionation on a Percoll density gradient. The amount of these HRP tracers in the lysosomal fraction became larger as the period of cell incubation was increased. Km values for uptake of HRP-anti-lamp-2, and LAP Fab' fragments were 0.74 and 0.62 microM, respectively, which were comparable to that of HRP-anti-lamp-1 Fab' (0.57 microM). The endocytic process of the two HRP-antibodies continued for an extended period in the cells exposed to the protein synthesis inhibitor, cycloheximide. Furthermore, we measured the transit times of HRP-anti-lamp-1, anti-lamp-2, and anti-LAP Fab' fragments from the cell surface to lysosomes.(ABSTRACT TRUNCATED AT 250 WORDS)
Akasaki
K
K
Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Hiroshima.
Fukuzawa
M
M
Kinoshita
H
H
Furuno
K
K
Tsuji
H
H
D000135:Acid Phosphatase / Q000276:immunology / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D015703:Antigens, CD; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D003078:Colchicine / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D004705:Endocytosis; D007124:Immunoenzyme Techniques; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007700:Kinetics; D008099:Liver / Q000648:ultrastructure*; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D051381:Rats
D015703:Antigens, CD; D007140:Immunoglobulin Fab Fragments; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D003513:Cycloheximide; D000135:Acid Phosphatase; D003078:Colchicine