Transport ofthe Lysosomal Membrane Glycoprotein 1gp120 (lgpA) to Lysosomes Does Not Require Appearance on the Plasma Membrane

We have used stably transfected CHO cell lines to characterize the pathway of intracellular transport of the 1gp120 (lgp--A) to lysosomes . Using several surface labeling and internalization assays, our results suggest that 1gp120 can reach its final destination with or without prior appearance on the plasma membrane . The extent to which lgp120 was transported via the cell surface was determined by two factors : expression level and the presence of a conserved glycine-tyrosine motif in the cytoplasmic tail . In cells expressing low levels of wild-type 1gp120, the majority of newly synthesized molecules reached lysosomes without becoming accessible to antibody or biotinylation reagents added extracellularly at 4°C. With increased expression levels, however, an increased fraction of transYSOSOMES contain a characteristic set of highly glycosylated membrane proteins . By cloning cDNAs from different species (Cha et al ., 1990 ; Chen et al ., 1988 ; Fambrough et al ., 1988 ; Granger et al ., 1990 ; Himeno et al ., 1989 ; Howe et al ., 1988 ; Noguchi et al ., 1989 ; Viitala et al ., 1988), they have been divided in two groups (lgpA and lgp-B) that share a high degree of homology in their overall structure and sequence (Kornfeld and Mellman, 1989) . Features common to all Igps include a large number of N-linked oligosaccharides in their luminal domain, a single transmembrane anchor, and a short 10-11-amino acid cytoplasmic tail . Despite their well-characterized biochemical properties, nothing is known about the functions of lgp's . Another important but as yet unresolved issue concerns the pathways taken by lgp's from the Golgi complex to late endosomes and lysosomes. Transport to lysosomes might occur after the delivery of newly synthesized lgp's to the plasma membrane and subsequent endocytosis. Alternatively, lgp's might be sorted intracellularly and reach endosomes and/or lysosomes without appearing on the cell surface . Several considerations suggest that either or both possibilities may occur. Kinetic studies of1gp120 (lgp --A) transport to lysosomes in normal rat kidney (NRK)' cells and mouse macrophages 1. Abbreviations used in this paper: LAP, lysosomal acid phosphatase ; MPR, manpose 6-phosphate receptor; NRK cells, normal rat kidney cells ; TGN, trans-Golgi network. © The Rockefeller University Press, 0021-9525/92/04/311/15 $2 .00 The Journal of Cell Biology, Volume 117, Number 2, April 1992 311-325 fected lgp120, as well as some endogenous lgp-B, appeared on the plasma membrane . The fraction of newly synthesized 1gp120 reaching the cell surface was also increased by mutations affecting the cytoplasmic domain tyrosine or glycine residues . A substantial fraction of both mutants reached the surface even at low expression levels . However, only the 1gpl20G-A7 mutant was rapidly internalized and delivered from the plasma membrane to lysosomes . Taken together, our results show that the majority of newly synthesized wild-type 1gpl20 does not appear to pass through the cell surface en route to lysosomes . Instead, it is likely that lysosomal targeting involves a saturable intracellular sorting site whose affinity for lgp's is dependent on a glycine-tyrosine motif in the 1gp120 cytoplasmic tail . suggested directed delivery of lgps to lysosomes without initial transport to the plasma membrane (Green et al ., 1987) . This conclusion was based on the observation that 1gp120 was transported from the Golgi complex to lysosomes as fast as plasma membrane proteins were transported from the Golgi complex to the cell surface ; thus, the initial cohort of newly synthesized lgp molecules reached lysosomes too rapidly to have involved obligatory transport through the cell surface. A similar conclusion was reached by cell fractionation for mouse lamp-1 (lgp-A) in 3T3 cells (D'Souza and August, 1986) . Conceivably, transport of lysosomal membrane components may follow the same pathway taken by newly synthesized lysosomal enzymes bound to the cationindependent mannose 6-phosphate receptor (MPR) . In this case, several considerations indicate that enzyme-receptor complexes leave the traps-Golgi network (TGN) in clathrincoated vesicles which deliver their contents to endosomes and, in turn, to lysosomes without reaching the cell surface (for review see Kornfeld and Mellman, 1989) . A similar finding for lgp's would imply that these molecules too possess a sorting determinant that directs their transport upon exit from the TGN. The alternative route, appearance on the cell surface followed by endocytosis, has been suggested to occur for at least a portion of the lgp molecules in chicken fibroblasts (LEP100, lgp-A ; Lippincott-Schwartz and Fambrough, 1986), primary rat hepatocytes (LGP107, lgp-A ; Furuno et al ., 1989a,ó), human leukemia cells (lamp-1, lgp-A ; Mane et on July 9, 2017 jcb.rress.org D ow nladed fom al ., 1989), and MDCK cells (AC17 antigen ; Nabi et al ., 1991) . Based on antibody binding, each of these studies found 2-3% of total lgp on the cell surface at steady state and that anti-lgp antibody could be internalized at 37 °C. Accordingly, these studies did not, however, determine the fraction ofnewly synthesized lgp that reached lysosomes via the plasma membrane . While recent pulse-chase experiments suggest that in MDCK cells the bulk of at least one lgp-like molecule appears at the basolateral plasmamembrane before reaching lysosomes, quantitative interpretation is limited by the fact that "surface appearance" was determined after antibody addition at 37°C and could not be distinguished from antibody binding in endosomes (Nabi et al ., 1991) . Transport via the cell surface to lysosomes has also been observed in stably transfected BHK cells overexpressing human lysosomal acid phosphatase (LAP), a soluble lysosomal enzyme that is transported as a membrane protein precursor (Braun et al ., 1989) . However, kinetic data seem to indicate that LAP, which is not a member of the lgpl20 family, is likely to reach lysosomes by a route distinct from lgp's . Whereas molecules such as 1gpl20 are transported to lysosomes with a t z of ti 30 min, LAP reaches lysosomes with a tut of 5-6 h and is recycled repeatedly between the plasma membrane and endosomes en route . A potential plasma membrane intermediate in lgp transport from the TUN to lysosomes is also indirectly implied by the fact that all lgp's, like many plasma membrane receptors (Davis et al ., 1986 ; Jing et al ., 1990; Lobel et al ., 1989), contain conserved cytoplasmic domain tyrosine residues that may serve as signals for rapid endocytosis (Kornfeld and Mellman, 1989 ; Ktistakis et al ., 1990) . Thus, lgp-A or LAP mutants in which this tyrosine was altered accumulate on the cell surface and cannot be internalized (Peters et al ., 1990 ; Williams and Fukuda, 1990) . While the failure of rapid endocytosis might account for the inability of these mutants to reach lysosomes, it is also possible that the conserved tyrosine residue plays a role in intracellular sorting, perhaps reflecting the accumulation of lgp's in clathrin-coated buds in the TGN. Interestingly, all lgp's also contain a conserved glycine residue on the amino-terminal side of the tyrosine; the significance of this residue for targeting to lysosomes is unknown . We have generated a series of stably transfected CHO cell lines selected for different expression levels of wild-type 1gp120 or various cytoplasmic domain mutants . Using several independent assays to selectively monitor the kinetics of surface appearance vs . internalization of newly synthesized lgp's, we have been able to better define the extent to which lgp's pass through the plasma membrane en route to lysosomes and the factors that regulate cell surface transport . We have found that under normal conditions, most of the lgp is sorted intracellularly and reaches lysosomes without appearing on the plasma membrane . However, intracellular sorting can be rendered less efficient, and the degree of surface transport increased, by amplifying the level oflgp expression or by cytoplasmic domain mutations that more severely affect targeting from the TGN than endocytosis . Materials and Methods Cell Culture and Transfection NRK fibroblasts were maintained in DME containing 5% FBS . CHO-KI The Journal of Cell Biology, Volume 117, 1992 were grown in a-MEM supplemented with 10% FBS . CHO cell lines deficient in dihydrofolate reductase, DUKX-B1 l and DG44, were obtained from Dr. L. Chasin (Columbia University, NY) . The untransfected mutant cells were maintained in a-MEM supplemented with nucleosides and 10% FBS . All cells were grown at 37°C in 5 % COZ in the presence of 100 U/ml penicillin and 100 mg/ml streptomycin . CHO cells were stably transfected with 10,ug plasmid DNA/10-cm tissue culture dish of 30% confluency by the calcium phosphate precipitation technique (Wigler et al ., 1979) . On day 2 after transfection, cells were dissociated with trypsin/EDTA and plated in selective medium containing 600 #<g/ml G418 (Gibco BRL, Gaithersburg, MD ; CHO-Kl) or in a-MEM without nucleosides supplemented with 10% dialyzed FBS (DUKX-BI1 and DG44). To amplify expression, DUKX-BI1 cells, transfected with pMT21 wild-type Igp120 DNA, were plated in selection medium containing 4 1+g/ml folic acid and 0.1 pM methotrexate (Lederle Parenterals, Inc., West Grove, PA) . Stable cell lines were obtained by subcloning of single colonies or by limiting dilution 14 d after transfection . For most experiments, cells were plated out 24-48 h