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© The Rockefeller University Press,
0021-9525/1997//1161 $5.00
The Journal of Cell Biology, Volume 137, Number 5,
, 1997 1161-1169
Article |
Different Steady State Subcellular Distributions of the Three Splice Variants of Lysosome-associated Membrane Protein LAMP-2 Are Determined Largely by the COOH-terminal Amino Acid Residue
The extensively glycosylated lysosome-associated membrane proteins (LAMP)-2a, b, and c are derived from a single gene by alternative splicing that produces proteins with differences in the transmembrane and cytosolic domains. The lysosomal targeting signals reside in the cytosolic domain of these proteins. LAMPs are not restricted to lysosomes but can also be found in endosomes and at the cell surface. We investigated the subcellular distribution of chimeras comprised of the lumenal domain of avian LAMP-1 and the alternatively spliced domains of avian LAMP-2. Chimeras with the LAMP-2c cytosolic domain showed predominantly lysosomal distribution, while higher levels of chimeras with the LAMP-2a or b cytosolic domain were present at the cell surface. The increase in cell surface expression was due to differences in the recognition of the targeting signals and not saturation of intracellular trafficking machinery. Site-directed mutagenesis defined the COOH-terminal residue of the cytosolic tail as critical in governing the distributions of LAMP-2a, b, and c between intracellular compartments and the cell surface.
Lysosome-associated membrane proteins (LAMPs)1 are major components of the lysosomal membrane. Two structurally related classes of LAMPs, LAMP-1 and -2, occur in birds and mammals, encoded by separate but evolutionally related genes. Both classes of LAMPs have a large, extensively glycosylated lumenal/extracellular domain, a single transmembrane domain, and a short cytosolic tail (for reviews see Fukuda, 1991; Peters and von Figura, 1994). Recently, multiple mRNAs have been identified for the LAMP-2 class (Hatem et al., 1995; Konecki et al., 1995). These mRNAs arise by alternative splicing of a single transcript and encode LAMP-2 molecules with different transmembrane and cytosolic domains (Gough et al., 1995).
The consensus targeting sequence, G-Y-X-X-hydrophobe, for delivery of the LAMPs to lysosomes resides in the cytosolic tail (Williams and Fukuda, 1990; Mathews, et al., 1992; Guarnier et al., 1993; Höning and Hunziker, 1995; Höning et al., 1996). LAMPs can reach the lysosome via two intracellular routes: a "direct" pathway from the trans-Golgi network either to the late endosomes or directly to lysosomes (Green et al., 1987; Harter and Mellman, 1992; Hunziker and Geuze, 1996), or an "indirect" pathway, which involves transport to early endosomes and the cell surface, where rounds of exocytosis and endocytosis may occur before delivery to lysosomes (LippincottSchwartz and Fambrough, 1986, 1987; Nabi et al., 1991; Carlsson and Fukuda, 1992; Mathews, et al., 1992; Akasaki et al., 1995). The signals for both direct and indirect delivery are contained within the targeting sequence.
The relative amounts of LAMPs expressed at the cell surface in the steady state appear to vary with cell type and physiological state. Many cells have very low levels of LAMPs at the cell surface; however, highly metastatic tumor cells (Saitoh et al., 1992), activated macrophages (Ho and Springer, 1983), strongly stimulated platelets (Febbraio and Silverstein, 1990; Silverstein and Febbraio, 1992), and retinoic acid-induced embryonal carcinoma cells (Amos and Lotan, 1990) show increased levels of LAMPs at the cell surface. LAMPs expressed at the cell surface carry complex oligosaccharide chains that bind to E-selectin (Sawada et al., 1994) and galaptin (Inohara and Raz, 1994; Woynarowska et al., 1994) and are substrates for cell surface β-1,4-galactosyltransferase (Maillet and Shur, 1993). Thus, cell surface LAMPs may be important mediators of cellular adhesion through interactions of their carbohydrate chains with carbohydrate receptors. Regulation of the subcellular distribution of LAMPs between the plasma membrane and the endocytic pathway may therefore be crucial in control of LAMP-mediated cell–cell interactions. Perhaps this is why two targeting routes are available to LAMPs. In the study reported here, we explored the relationship between LAMP targeting signals and subcellular distribution.
The LAMP-2 splice variants have different transmembrane domains and cytosolic tails. These differences include different amino acids within the COOH-terminal G-Y-X-X-hydrophobe sequence that participates in protein targeting. Previously we showed that the lysosomal targeting signal in the cytosolic tail of each of the LAMP-2 variants is competent for the delivery of chimeric LAMP proteins to lysosomes (Hatem et al., 1995). In this study we tested the hypothesis that the differences in LAMP-2 targeting signals affect the steady-state distribution of the LAMP-2 variants in cells. We report that the steady state levels of the LAMP-1/LAMP-2 chimeras at the cell surface are different and that these differences are correlated with differences in the LAMP-2 COOH-terminal targeting signals.
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Materials and Methods |
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Cell Lines and Culture
Plasmids were introduced into mouse L cells using Lipofectin (Life Technologies, Inc., Gaithersburg, MD) with Optimem medium (5 µg DNA/ 60-mm dish of 60% confluent cells). Stable cell lines were selected and maintained in Dulbecco's minimal essential medium (DMEM) with 10% fetal calf serum and 1% gentamycin, supplemented with 400 µg/ml G418. Expression of chimeric LAMPs was induced by treatment of the transfected cells with 1 mM butyrate for 48 ± 2 h, and the subcellular distribution of LAMPs in individual cell lines (designated with a number) was characterized. To minimize the possibility that observed differences in LAMP distribution were due to peculiarities of individual cell lines, entire dishes of nonclonal transfected L cells (designated "pool") were also assayed as a single sample. Individual cell lines were heterogeneous in terms of expression level per cell and the number of positive cells, as judged by immunofluorescence microscopy.
Chimera Detection and Quantification
The distribution of the LAMP chimeras was determined using a monoclonal antibody (mAb-CV24) against the lumenal domain of avian LAMP-1, formerly called LEP100 (Lippincott-Schwartz and Fambrough, 1987). This antibody recognizes the lumenal/extracellular domain and will bind to LAMP chimeras expressed on the surface of intact cells. Also, this antibody can be radiolabeled with 125I without loss of activity, permitting direct detection and quantification of the amount of the chimera expressed by the transfected cells.
Immunofluorescent labeling of permeabilized cells was performed, after fixation in 1% formaldehyde, with 2 µg/ml mAb-CV24 in Hanks' balanced salt solution, 20 mM Tris-Cl, pH 7.5, and 2% horse serum (H/T/HS) supplemented with 0.1% saponin. The primary antibody was detected with FITC-conjugated goat anti–mouse secondary antibody (Kirkegaard & Perry, Gaithersburg, MD). Immunofluorescent labeling of intact cells was performed, after fixation in 1% formaldehyde, with 2 µg/ml FITC-conjugated mAb-CV24 in H/T/HS without saponin.
Iodinated antibody binding was performed on permeabilized and intact fixed cells. mAb-CV24 (100 µg) was iodinated by the Iodogen method (Salicinski et al., 1981). Binding was performed, after fixation in 1% formaldehyde, with 2 µg/ml 125I–mAb-CV24 in H/T/HS with or without 0.1% saponin. Nonspecific binding was measured in the presence of 25-fold excess cold mAb-CV24 in the presence and absence of saponin. Each sample was assayed in triplicate. Specific binding was calculated by subtracting the nonspecific counts per minute from total counts per minute for both permeabilized and intact conditions. The fraction of LAMP molecules present at the cell surface was calculated by dividing the specific counts per minute of 125I–mAb-CV24 bound in the absence of saponin by the specific counts per minute bound in the presence of saponin.
Antibody Internalization Assay
The internalization of LAMP molecules expressed on the cell surface was estimated by measuring the rate at which 125I-labeled mAb-CV24 bound to LAMP molecules at the cell surface became resistant to removal from the surface by acid stripping. Stable cell lines were plated in 6-well plates at 5 x 105 cells/well and induced with butyrate for 48 h. Cells were cooled on wet ice, washed once with cold H/T/HS, and incubated for 4 h with 2 µg/ml 125I–mAb-CV24 in H/T/HS. The cells were washed in 2 baths of ice cold H/T/HS for 15 min each to remove unbound antibody. Cells were either kept on wet ice or incubated at 37°C in prewarmed Hepes-buffered growth medium for the indicated times. The medium was collected. The surface antibody was stripped with ice cold 0.1 M acetic acid/1.5 M NaCl for 30 min on wet ice and collected. Finally, the cells were extracted in 1 N NaOH at room temperature to determine cell-associated antibody. The media, acetic acid solutions, and NaOH extracts were counted in a 
counter. Nonspecific binding was determined from cultures handled identically, except that a 25-fold excess of unlabeled mAb-CV24 was included in the 125I–mAb-CV24 binding solution. After corrections for nonspecific binding, the percent of bound antibody that was internalized was calculated by dividing the NaOH-extracted counts by the total counts present in the medium, acetic acid, and NaOH extract.
Quantification of Endogenous Mouse LAMP-1
Endogenous mouse LAMP-1 was detected with the monoclonal antibody 1D4B, a gift from Dr. J.T. August (The Johns Hopkins University School of Medicine, Baltimore, MD). For immunofluorescence microscopy and iodinated antibody binding, the cells were processed as described above for detection and quantification of avian LAMP chimeras. The fraction of endogenous mouse LAMP-1 expressed at the cell surface was determined from binding experiments with 125I-labeled mAb-1D4B; the binding experiments and calculations were analogous to those described above for quantifying the distribution of the avian LAMP chimeras. mAb-1D4B was used at a concentration of 10 µg/ml for both immunofluorescence microscopy and iodinated antibody binding assays. The binding assays were performed in two independent experiments with duplicate samples for total and nonspecific binding in the presence and absence of 0.1% saponin.
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Results |
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Although each of the LAMP-1/LAMP-2 chimeras was capable of being targeted to lysosomes (Hatem et al., 1995), differences in their levels of expression at the cell surface were readily apparent in direct immunofluorescent labeling of intact cells (Fig. 1). Cells expressing the L/2c/2c chimera showed negligible levels of surface immunofluorescent labeling, to the extent that it was difficult to identify positive expressing cells by direct immunofluorescence labeling without permeabilization (Fig. 1 f). Cells expressing the L/2a/2a chimeras were more variable in surface expression; some cell lines contained easily identifiable, positive cells (lines 19 and 20) while in other lines the cells were considerably fainter in their immunofluorescence (line 10) without permeabilization. Cells expressing the L/2b/2b chimera showed the highest levels of surface expression (Fig. 1 d).
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50,000 to 400,000. In one extreme case, a line expressing 1 x 106 molecule/cell of a chimera with the LAMP-2c cytosolic tail had only 2% of the molecules at the cell surface. Thus, overexpression resulting in mistargeting of molecules to the plasma membrane was not a complication in our study.
The Cytosolic Tail Determines Subcellular Distribution
To determine whether the differences in subcellular distribution of the various LAMP chimeras were due to differences in the transmembrane domain or in the cytosolic tail, "tail switch" chimeras (L/2b/2c and L/2c/2b) were constructed, and their subcellular distributions in transfected mouse L cells were determined. As shown in Fig. 5, cells expressing chimeras with the LAMP-2c cytosolic tail showed a very low level immunofluorescent labeling at the cell surface of intact cells while showing intense labeling after permeabilization (Fig. 5, a and b). Cells induced to express the chimeras with the LAMP-2b cytosolic tail showed high levels of surface expression, evidenced by immunofluorescent labeling of intact cells (Fig. 5, c and d). Quantitatively, the L/2c/2b chimeras were expressed at 3.3-fold higher levels at the cell surface than the L/2b/2c chimeras (Table III).
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The COOH-terminal Residue Influences LAMP Subcellular Distribution
To assess which amino acids within the cytosolic tails of the LAMP-2 molecules are critical in conferring high or low levels of surface expression, point mutations were created in the cytosolic tails of the L/2b/2b and L/2c/2c chimeras. Amino acids in the cytosolic tail of LAMP-2b were replaced with amino acids present in the LAMP-2c tail and vice versa. L cells expressing the chimeras containing the point mutations were analyzed by immunofluorescence microscopy (Fig. 6) and quantitative antibody binding (Table IV). The first mutation L/2b/2b R391Y was outside the G-Y-X-X-hydrophobe targeting sequence. This mutant showed high levels of surface expression similar to its parent chimera L/2b/2b, suggesting that this residue was not part of the signal for efficient lysosomal targeting of LAMP-2c (Fig. 6 b, and Table IV). Two mutations were created in the G-Y-X-X-hydrophobe sequence of LAMP-2b S396T and V397L. The L/2b/2b S396T mutant showed high levels of surface expression, indicating that this residue was not sufficient to confer more efficient lysosomal targeting information to the LAMP-2b cytosolic tail (Fig. 6 d, and Table IV). However, the L/2b/2b V397L mutant showed remarkably lower surface expression, similar to that seen with the L/2c/2c chimera, suggesting that the COOH-terminal residue is important for the high efficiency of lysosomal targeting of LAMP-2c (Fig. 6 f, and Table IV). The double mutant containing the S396T and V397L mutations behaved as the V397L mutant with low expression at the cell surface (Table IV). To confirm that the COOH-terminal residue could dictate the relative levels of surface expression of the LAMP-2 variants, one additional chimera (L/2c/2c L396V) was generated which changes the COOH-terminal leucine of LAMP-2c to a valine, the COOH-terminal residue of LAMP-2b. (Note that LAMP-2c is shorter by one amino acyl residue than LAMP-2b; position 396 is the penultimate residue in LAMP-2b but the COOH-terminal residue in LAMP-2c.) This chimera was expressed at high levels at the cell surface (Fig. 6 h, and Table IV), consistent with the COOHterminal residue being the essential component of the signal for dictating relative surface expression of the LAMP2 variants in the steady state.
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6 min, indicating that the cytosolic tails of these two chimeras can interact with the molecular machinery that mediates endocytosis. These cells essentially cleared the surface of the chimera during the 60 min incubation at 37°C, suggesting either that the endosomal pool of chimera molecules was much larger than the surface pool or that most of the endocytosed molecules were not being recycled back to the cell surface. Cells expressing chimera L/2b/2b or L/2c/2c L396V did not internalize iodinated mAb-CV24 efficiently, indicating that valine in the COOH-terminal position impaired the ability of the cytosolic tail to be recognized by the endocytic machinery. We suggest that the relatively high levels of surface expression of the L/2b/2b and L/2c/2c L396V chimeras are attributable, at least in part, to drastically decreased rates of endocytosis.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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As the results of this study demonstrate, LAMP-1/ LAMP-2 chimeras show differences in steady state distribution that are attributable to amino acid differences in the cytosolic tail domains of the splice variants of LAMP-2. Cells expressing LAMP-1/LAMP-2c showed very low levels of surface expression similar to the levels of LAMP-1, as described for primary cultures of chicken embryo fibroblasts (Lippincott-Schwartz and Fambrough, 1986) and L cells (Mathews et al., 1992) and MDCK cells transfected with chicken LAMP-1 (Nabi et al., 1991). Cells expressing LAMP-1/LAMP-2a or LAMP-1/LAMP-2b showed much higher levels of surface expression, suggesting that the steady state distribution may be influenced by the different cytosolic tail sequences. Chimeras in which the cytosolic tails of LAMP-2b and c were switched and chimeras with point mutations in the cytosolic tails of LAMP-2b and c showed different levels of surface expression, demonstrating that differences in subcellular distribution of LAMP-2 splice variants can be attributed to differences in their cytosolic tails. These results suggest that the LAMP-2 splice variants may have quantitatively different subcellular distributions in vivo and that the function of LAMP-2 molecules may be related, at least in part, to their expression at the plasma membrane.
It is possible that each LAMP has multiple functions and that different functions involve different compartments of the cell. Additionally, it is possible that each LAMP variant plays a unique role in cells that express it. Within the lysosome, the extensively glycosylated LAMPs that accumulate in high abundance, such as LAMP-1 and -2c, may provide a barrier to prevent degradation of the lysosomal membrane lipid bilayer and the lysosomal membrane proteins that are important for lysosomal function. On the other hand, the expression of LAMP molecules at high levels in the plasma membrane, such as occurs with LAMP-2a and b, as well as the redistribution of LAMPs to the cell surface in certain situations, may provide opportunities for interactions between LAMPs and receptors on other cells or in the extracellular matrix. Such interactions could influence cellular adhesion or participate in other intercellular events. Less likely, but not excluded by present information, LAMPs could be binding to extracellular components which are then endocytosed together with the LAMP molecules, the LAMP molecules essentially serving as receptors in receptor-mediated endocytosis.
Mechanistically, the differences in subcellular distribution may reflect differences in the abilities of the cytosolic tail targeting sequences to be recognized by intracellular trafficking machinery. The G-Y-X-X-hydrophobe sequence is believed to be recognized by adaptin complexes, AP-1 at the trans-Golgi network and AP-2 at the plasma membrane (Ohno et al., 1995; Höning et al., 1996). Differences within this G-Y-X-X-hydrophobe consensus sequence, such as those seen among the LAMP-2 variants, may result in different affinities for the adaptin complexes at various steps in the biosynthetic/targeting pathway. Consistent with this possibility, the chimeras with high surface expression, such as L/2b/2b and L/2c/2c L396V, were found to be deficient in their ability to be endocytosed from the cell surface, suggesting that the AP-2 complex may not efficiently recognize LAMP cytosolic tails that have a valine in the COOH-terminal position. Such an inference is congruent with observations of Ohno et al. (1995) that when the targeting sequence of TGN38 (SDYQRL) was made COOH terminal, replacing the COOH-terminal leucine with valine greatly decreased its interaction with the AP-2 m subunit and to a lesser degree decreased its interaction with the AP-1 m subunit, assessed in yeast two hybrid assays. Differences in recognition of LAMP cytosolic tails by intracellular trafficking machinery at the level of the trans-Golgi network, the plasma membrane, and the endosomal compartment may also contribute to the differences in subcellular distribution of LAMPs. Whatever the critical sorting sites and molecular mechanisms, the identity of the COOH-terminal residue provides an important signal to determine the subcellular distribution of the LAMP-2 variants.
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Acknowledgments |
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This work was supported by National Institutes of Health grant NS23241 awarded to Dr. D.M. Fambrough.
Submitted: 27 November 1996
Revised: 12 February 1997
1. Abbreviation used in this paper: LAMP, lysosome-associated membrane protein.
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