The kinetics of mannose 6-phosphate receptor trafficking in the endocytic pathway in HEp-2 cells: the receptor enters and rapidly leaves multivesicular endosomes without accumulating in a prelysosomal compartment - PubMed (original) (raw)

The kinetics of mannose 6-phosphate receptor trafficking in the endocytic pathway in HEp-2 cells: the receptor enters and rapidly leaves multivesicular endosomes without accumulating in a prelysosomal compartment

J Hirst et al. Mol Biol Cell. 1998 Apr.

Free PMC article

Abstract

We have previously shown that in HEp-2 cells, multivesicular bodies (MVBs) processing internalized epidermal growth factor-epidermal growth factor receptor complexes mature and fuse directly with lysosomes in which the complexes are degraded. The MVBs do not fuse with a prelysosomal compartment enriched in mannose 6-phosphate receptor (M6PR) as has been described in other cell types. Here we show that the cation-independent M6PR does not become enriched in the endocytic pathway en route to the lysosome, but if a pulse of M6PR or an M6PR ligand, cathepsin D, is followed, a significant fraction of these proteins are routed from the trans-Golgi to MVBs. Accumulation of M6PR does not occur because when the ligand dissociates, the receptor rapidly leaves the MVB. At steady state, most M6PR are distributed within the trans-Golgi and trans-Golgi network and in vacuolar structures distributed in the peripheral cytoplasm. We suggest that these M6PR-rich vacuoles are on the return route from MVBs to the trans-Golgi network and that a separate stable M6PR-rich compartment equivalent to the late endosome/prelysosome stage does not exist on the endosome-lysosome pathway in these cells.

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Figures

Figure 1

Figure 1

Double label of M6PR and EGF en route to the lysosome. Cells were incubated with EGF-TxR (red) for 10–60 min at 37°C and were then immunolabeled for M6PR (green). After 10 min at 37°C, EGF-TxR is in vacuoles distributed throughout the peripheral cytoplasm (A). With further incubation at 37°C for 25 (B), 45 (C), and 60 min (D), EGF-TxR accumulates in the juxtanuclear region of the cell (large arrows). At all time points most of the elements labeled with EGF and M6PR are discrete and separate. Small arrows indicate the rare occasions of coincidence between EGF-TxR and M6PR.

Figure 2

Figure 2

Triple label showing M6PR in relation to markers of the _trans_-Golgi and endosome. Cells transiently transfected with ST-HRP were incubated with TF-FITC (green) for 60 min at 37°C and were then immunolabeled for M6PR (red) and HRP (blue). Merged images are shown in A and B, and single images of the cell shown in B are shown in C (M6PR) and D (ST-HRP). M6PR is found in the _trans_-Golgi region, in which _trans_-Golgi elements that also contain ST-HRP are stained magenta, and in vacuoles in the peripheral cytoplasm. The majority of the peripheral M6PR-positive vacuoles stain red (arrows) and so do not contain either ST-HRP or TF-FITC. Only a few M6PR-positive vacuoles contain TF (stained yellow), and TF-FITC is not detectable in the _trans_-Golgi.

Figure 3

Figure 3

Trafficking of newly synthesized M6PR through MVBs. To measure the rate of appearance of newly synthesized M6PR in MVBs, HEp-2 cells were pulsed with 35S-labeled amino acids for 15 min at 37°C, washed, and then maintained at 20°C for 4 h. Anti-TR-gold was added for the last hour at 20°C before being transferred to 37°C for up to 30 min in the continued presence of anti-TR-gold. Gold-loaded MVBs were then isolated as described in MATERIALS AND METHODS. The amount of newly synthesized M6PR in the isolated MVB fractions was determined by immunoprecipitation and expressed as a percentage of the total loaded on the fractionation gradient (% PNS). Results are means ± SE of four observations.

Figure 4

Figure 4

Trafficking of newly synthesized CD through the MVBs. To measure the rate of appearance of newly synthesized CD in MVBs, HEp-2 cells were pulsed with 35S-labeled amino acids for 15 min at 37°C, washed, and incubated for 20 min at 37°C and then maintained at 20°C for 4 h. Anti-TR-gold was added for the last hour at 20°C before being transferred to 37°C for up to 30 min in the continued presence of anti-TR-gold. Gold-loaded MVBs were then isolated as described in MATERIALS AND METHODS. The amount of newly synthesized CD in the isolated MVB fractions was determined by immunoprecipitation and expressed as a percentage of the total loaded on the fractionation gradient (% PNS). Results are means ± SE of three observations.

Figure 5

Figure 5

The effect of tunicamycin on trafficking of CD through MVBs. Cells were preincubated with tunicamycin (10 μg/ml) for 2 h at 37°C, and then pulsed with 35S-labeled amino acids for 15 min at 37°C, washed, and incubated for 20 min at 37°C and then maintained at 20°C for 4 h. Anti-TR-gold was added for the last hour at 20°C before transfer to 37°C for up to 60 min in the continued presence of anti-TR-gold. Gold-loaded MVBs were then isolated as described in MATERIALS AND METHODS. The amount of newly synthesized CD in the isolated MVB fractions was determined by immunoprecipitation and expressed as a percentage of the total loaded on the fractionation gradient (% PNS).

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References

    1. Aniento F, Emans N, Griffiths G, Gruenberg J. Cytoplasmic dynein-dependent vesicular transport from early to late endosomes. J Cell Biol. 1993;123:1373–1387. - PMC - PubMed
    1. Beardmore J, Howell KE, Miller K, Hopkins CR. Isolation of an endocytic compartment from A431 cells using a density modification procedure employing a receptor-specific monoclonal antibody complexed with colloidal gold. J Cell Sci. 1987;87:495–506. - PubMed
    1. Bomsel M, Parton R, Kutznetsov SA, Schroer TA, Gruenberg J. Microtubule- and motor-dependent fusion in vitro between apical and basolateral endocytic vesicles from MDCK cells. Cell. 1990;62:719–731. - PubMed
    1. Brown WJ, Goodhouse J, Farquhar MG. Mannose 6-phosphate receptors for lysosomal enzymes cycle between the Golgi complex and endosomes. J Cell Biol. 1986;103:1235–1247. - PMC - PubMed
    1. Campbell C, Rome LH. Coated vesicles from rat liver and calf brain contain lysosomal enzymes bound to mannose 6-phosphate receptors. J Biol Chem. 1983;258:13347–13352. - PubMed

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