Abstract

The lysosome has been chosen as a model to study the exchange of native membrane proteins within an organelle population. Heterologous lysosomes were brought together by vesicular stomatitis virus-mediated cell fusion. The distribution of lysosomal membrane protein was visualized by indirect immunofluorescence using species-specific monoclonal antibody. LAMP-2, a mouse lysosomal membrane protein, and HLAMP-B, a human lysosomal membrane protein, were found to transfer to Chinese hamster ovary cell sucrosomes (sucrose-swollen lysosomes). This transfer occurred in the presence of cycloheximide. The exchange of LAMP-2 and LIMP I, a rat lysosomal membrane protein, was observed between native lysosomes in a mouse (3T3)-rat (normal rat kidney) cell fusion. Extensive transfer/exchange was observed within 1.5-2 hr postfusion, which is consistent with the kinetics of endocytic content exchange between lysosomes. Both membrane protein and content transfer between lysosomes were inhibited by nocodazole, a disrupter of microtubules, as was endocytic delivery to sucrose-swollen lysosomes. In the presence of nocodazole, tubular lysosomes disappeared. Both tubular lysosomes and microtubules may be important for the transfer/exchange. The interspecies cell fusion/monoclonal antibody approach developed here should be readily applicable to determining if membrane protein exchange is a property of other organelles such as Golgi apparatus and mitochondria.