DIFFERENCES IN THE ENDOSOMAL DISTRIBUTIONS OF THE 2 MANNOSE 6-PHOSPHATE RECEPTORS

Multiple immunolabeling of cryosections was performed to compare the s ubcellular distributions of the two mannose 6-phosphate receptors (MPR s) involved in the intracellular targeting of lysosomal enzymes: the c ation-dependent (CD) and cation-independent (CI) MPR. In two cell type s, 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 tw o receptors at the same sites: the trans-Golgi reticulum (TGR), endoso mes, electron-dense cytoplasmic vesicles, and the plasma membrane. In the TGR the two receptors colocalized and were concentrated to the sam e extent in the same HA I-adaptor positive coated buds and vesicles. E ndosomes were identified by the presence of exogenous tracers. The two MPR codistributed to the same endosomes, but semiquantitative analysi s showed a relative enrichment of the CI-MPR in endosomes containing m any internal vesicles. Two endosomal subcompartments were discerned, t he central vacuole and the associated tubules and vesicles (ATV). We f ound an enrichment of CD-MPR over CI-MPR in the ATV. Lateral segregati on of the two receptors within the plane of membranes was also detecte d 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 concen trated in different subpopulations of endosomal ATV. The small GTP-bin ding protein rab4, which has been shown to mediate recycling from endo somes to the plasma membrane, was localized at the cytosolic face of m any endosomal ATV. Quantitative analysis of double-immunolabeled cells revealed only a limited codistribution of the MPRs and rab4 in ATV. T hese data suggest that the two MPRs exit the TGR via the same coated v esicles, but that upon arrival in the endosomes CD-MPR is more rapidly than CI-MPR, segregated into ATV which probably are destined to recyc le MPRs to TGR.