Differences in the endosomal distributions of the two mannose 6-phosphate receptors.
Multiple immunolabeling of cryosections was performed to compare the subcellular distributions of the two mannose 6-phosphate receptors (MPRs) involved in the intracellular targeting of lysosomal enzymes: the cation-dependent (CD) and cation-independent (CI) MPR. In two cell types, the human hepatoma cell line HepG2 and BHK cells double transfected with cDNA's encoding for the human CD-MPR and CI-MPR, we found the two receptors at the same sites: the trans-Golgi reticulum (TGR), endosomes, electron-dense cytoplasmic vesicles, and the plasma membrane. In the TGR the two receptors colocalized and were concentrated to the same extent in the same HA I-adaptor positive coated buds and vesicles. Endosomes were identified by the presence of exogenous tracers. The two MPR codistributed to the same endosomes, but semiquantitative analysis showed a relative enrichment of the CI-MPR in endosomes containing many internal vesicles. Two endosomal subcompartments were discerned, the central vacuole and the associated tubules and vesicles (ATV). We found an enrichment of CD-MPR over CI-MPR in the ATV. Lateral segregation of the two receptors within the plane of membranes was also detected on isolated organelles. Double immunolabeling for the CD-MPR and the asialoglycoprotein receptor, which mainly recycles between endosomes and the plasma membrane, revealed that these two receptors were concentrated in different subpopulations of endosomal ATV. The small GTP-binding protein rab4, which has been shown to mediate recycling from endosomes to the plasma membrane, was localized at the cytosolic face of many endosomal ATV. Quantitative analysis of double-immunolabeled cells revealed only a limited codistribution of the MPRs and rab4 in ATV. These data suggest that the two MPRs exit the TGR via the same coated vesicles, but that upon arrival in the endosomes CD-MPR is more rapidly than CI-MPR, segregated into ATV which probably are destined to recycle MPRs to TGR.
Klumperman
J
J
Department of Cell Biology, Utrecht University, The Netherlands.
Hille
A
A
Veenendaal
T
T
Oorschot
V
V
Stoorvogel
W
W
von Figura
K
K
Geuze
H J
HJ
D000818:Animals; D037263:Asialoglycoprotein Receptor; D002451:Cell Compartmentation; D006224:Cricetinae; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism*; D008854:Microscopy, Electron; D017527:Receptor, IGF Type 2 / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014168:Transferrin / Q000378:metabolism; D014407:Tumor Cells, Cultured; D020695:rab4 GTP-Binding Proteins
D037263:Asialoglycoprotein Receptor; D017527:Receptor, IGF Type 2; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; D014168:Transferrin; D019204:GTP-Binding Proteins; D020695:rab4 GTP-Binding Proteins