ROUTING OF A SECRETORY PROTEIN TO THE ENDOCYTIC COMPARTMENT IN TRANSFECTED MADIN-DARBY CANINE KIDNEY-CELLS

Transfected Madin Darby canine kidney (MDCK) cells (3A) expressing hum an growth hormone (hGH) contain twice as many Golgi stacks as untransf ected cells. How MDCK cells, lacking a regulated pathway, deal with (o ver)expression of a protein hormone, or any exogenous protein, has not been examined in detail. Since hGH constituted 10% of total secreted proteins, it was not apparent why Golgi amplification was needed, unle ss some enters a nonsecretory compartment. Studies were undertaken to determine hGH fate. By using an inhibitor of protein synthesis, or by analyzing pulse labeled immunoprecipitated hGH, 20-30% of hGH was show n to remain intracellular even after 4 h. That portion might be locali zed in the endosome/lysosome compartment, because it is post-Golgi. Im munoelectron microscopy with antibodies against hGH, clathrin, and cat hepsin D demonstrated clathrin and hGH colocalized, as did hGH and cat hepsin D. The latter were found in large vesicles, but no hGH appeared in lysosomes, due to its degradation. Analysis of isolated lysosome/e ndosomes revealed vesicles containing both hGH and cathepsin D, but mo re containing only cathepsin D. Endocytosis studies suggested the 3A b asolateral endosomal compartment may be more capacious than normal. Th us, 3A Golgi amplification resulted in an expanded endosome compartmen t to accommodate secretory protein (over)expression.