Kinetic evidence that newly-synthesized endogenous lysosome-associated membrane protein-1 (LAMP-1) first transits early endosomes before it is delivered to lysosomes

After de novo synthesis of lysosome-associated membrane proteins (LAMPs), they are sorted in the trans-Golgi network (TGN) for delivery to lysosomes. Opposing views prevail on whether LAMPs are targeted to lysosomes directly, or indirectly via prelysosomal stages of the endocytic pathway, in particular early endosomes. Conflicting evidence is based on kinetic measurements with too limited quantitative data for sufficient temporal and organellar resolution. Using cells of the mouse macrophage cell line, P338D(1), this study presents detailed kinetic data that describe the extent of, and time course for, the appearance of newly-synthesized LAMP-1 in organelles of the endocytic pathway, which had been loaded selectively with horse-radish peroxidase (HRP) by appropriate periods of endocytosis. After a 5-min pulse of metabolic labelling, LAMP-1 was trapped in the respective organelles by HRP-catalyzed crosslinking with membrane-permeable diaminobenzidine (DAB). These kinetic observations provide sufficient quantitative evidence that in P338D(1) cells the bulk of newly-synthesized endogenous LAMP-1 first appeared in early endosomes, before it was delivered to late endosomes and lysosomes about 25 min later.