Lysosomes can fuse with a late endosomal compartment in a cell-free system from rat liver.

Abstract

Abstract. The passage of pulse doses of asialoglycoproteins through the endosomal compartments of rat liver hepatocytes was studied by subcellular fractionation and EM. The kinetics of disappearance of radiolabeled asialofetuin from light endosomes prepared on Ficoll gradients were the same as the kinetics of disappearance of asialoorosomucoid-horse radish peroxidase reaction products from intracellular membrane-bound structures in the blood sinusoidal regions of hcpatocytes. The light endosomes were therefore identifiable as being derived from the peripheral early endosomc compartment. In contrast, the labeling of dense endosomes from the middle of the Ficoll gradient correlated with EM showing large numbers of reaction product-containing structures in the nonsinusoidal parts of the hepatocytc.In ceil-free, postmitochondrial supcrnatants, we have previously observed that dense cndosomes, but not light endosomcs, interact with lysosomcs. Cell-free interaction between isolated dense endosomes and lysosomes has now been reconstituted and analyzed in three ways: by transfer of radiolabclcd ligand from endosomal to lysosomal densities, by a fluorescence dequenching assay which can indicate membrane fusion, and by measurement of content mixing. Maximum transfer of radiolabel to lysosomal densities required ATP and GTP plus cytosolic components, including N-cthylmaleimide-sensitive factor(s). Dense endosomes incubated in the absence of added lysosomcs did not mature into vesicles of lysosomal density. Content mixing, and hence fusion, between endosomes and lysosomcs was maximal in the presence of cytosol and ATP and also showed inhibition by N-ethylmaleimide. Thus, we have demonstrated that a fusion step is involved in the transfer of radiolabcled ligand from an isolated endosome fraction derived from the nonsinusoidal regions of the hepatocyte to preexisting lysosomcs in a ceU-frcc system. U NDERSTANDING of vesicular traffic pathways in the cell and their control has increased markedly in recent years, very largely as a result of the development of cell-free systems which permit identification of the proteins required. Components which operate at many steps on the secretory pathway have thus been characterized and shown to include GTP-binding proteins (Novick and Brcnnwald, 1993), N-ethylmaleimide (NEMy-sensitive factor, its soluble attachment proteins and, most recently, receptor proteins which confer specificity between vesicle and target (Sollner et al., 1993; lhkizawa and Malhotra, 1993 1. Abbreviations used in this paper: AMP-PNP, 5'-adenylylimidodiphosphate; ASE asialofetuin; ASOR-HRP, asialoorosomucoid-horse radish peroxidase conjugate; bplgA, biotinylated polymeric IgA; NEM, N-ethylmaleimide; RI8, octadecylrhodamine/~-chloride; RIPA, 1% sodium, 1% Triton X-100, 0.2% NP-40, 0.15 M NaCI, 0.05 M Tris, pH 7.5; STM, 0.25 M sucrose, 10 mM N-tris 01ydroxymethyl)-methyl-2-aminoethane sulphonic acid and I mM Mg 2+ (pH 7.4). Smythe and Warren, 1991; Schmid, 1993). In vivo it is known that hepatocytes internalize ligand...

In vitro endosome‐lysosome transfer of dephosphorylated EGF receptor and Shc in rat liver

“…As with asialofetuin, GalB-SA, glucagon and insulin [11,23,24], the cell-free transfer of EGF-EGFR complexes occurred from endosomes isolated at a late stage of endocytosis (time of killing 10 min ; [7,20]) and required the addition of cytosol and an ATP-regenerating system [11,23]. The endosome-lysosome transfer was accompanied by increased low molecular weight degradation products of EGF at the upper part of the gradient.…”

Lysosome Associated Membrane Protein 1 (Lamp1) Traffics Directly from the TGN to Early Endosomes

“…Early endosomes form the acceptor compartment in vitro Susceptible proteins delivered to lysosomes in vitro are rapidly degraded (37). Thus the stability of YAL shown in Figure 6B suggests that lysosomes are not an acceptor compartment for TGN-derived YAL in this assay.…”

Rab7: A Key to Lysosome Biogenesis

“…This model implies that, in addition to heterotypic fusions between late endosomes and lysosomes in the perinuclear region, there could also be continuous exchange within the lysosomal vesicle population (homotypic fusion). Indeed, evidence for fusions and bidirectional traffic of soluble material between lysosomes and late endosomes has been reported (Jahraus et al, 1994;Mullock et al, 1994;van Deurs et al, 1995;Futter et al, 1996;Storrie and Desjardins, 1996;Bright et al, 1997;Mullock et al, 1998).…”

“…This model implies that, in addition to heterotypic fusions between late endosomes and lysosomes in the perinuclear region, there could also be continuous exchange within the lysosomal vesicle population (homotypic fusion). Indeed, evidence for fusions and bidirectional traffic of soluble material between lysosomes and late endosomes has been reported (Jahraus et al, 1994;Mullock et al, 1994;van Deurs et al, 1995;Futter et al, 1996;Storrie and Desjardins, 1996;Bright et al, 1997;Mullock et al, 1998).Several different molecules are part of the machinery responsible for vesicle docking and fusion, Rab GTPases and SNAREs being among the best studied (Rothman and Warren, 1994;Denesvre and Malhotra, 1996;Pfeffer, 1996Pfeffer, , 1999Olkkonen and Stenmark, 1997;Mayer, 1999;Waters and Pfeffer, 1999). Rab proteins are important regulators of membrane traffic on the biosynthetic and endocytic pathways (Pfeffer, 1992;Zerial and Stenmark, 1993;Novick and Zerial, 1997;Olkkonen and Stenmark, 1997;Martinez and Goud, 1998;Chavrier and Goud, 1999;Pfeffer, 1999).…”