THE CARBOXY-TERMINAL REGION OF HUMAN LIPOPROTEIN-LIPASE IS NECESSARY FOR ITS EXIT FROM THE ENDOPLASMIC-RETICULUM

Certain missense substitutions on the human lipase (hLPL) gene produce mutated proteins that are retained in different compartments along th e secretory pathway. The purpose of the present study was to elucidate whether the C-terminal domain of the hLPL molecule could be important for secretion. We constructed by site-directed mutagenesis three carb oxy-terminal mutated (F-388-->Stop, K-428-->Stop and K-441-->Stop) hLP L cDNAs that were expressed in COS1 cells, Immunoblotting of cell extr acts showed that all three constructs led to similar levels of protein . Both wild type (WT) hLPL and the truncated K-441-->Stop hLPL were se creted to the extracellular medium, and presented a similar intracellu lar distribution pattern as shown by immunofluorescence. Neither F-388 -->Stop nor K-428-->Stop hLPL protein was detected in cell medium. Imm unofluorescence experiments showed that both truncated hLPL were retai ned within an intracellular compartment, which became larger. Double i mmunofluorescence analysis using antibodies against LPL and antiprotei n disulfide isomerase as a marker showed that the truncated K-428-->St op hLPL was retained within the rough endoplasmic reticulum. This trun cated protein was not found in other compartments in the secretory pat hway, such as Golgi complex and lysosomes, indicating that it did not exit the endoplasmic reticulum. Further analysis of the C-terminal reg ion of tile LPL molecular model showed both that F-388-->Stop and K-42 8-->Stop hLPL truncated proteins are highly hydrophobic. As retention of secretory proteins in the rough endoplasmic reticulum is a quality control mechanism of the secretory pathway, Eve conclude that the C-te rminal domain of hLPL is critical for correct intracellular processing of the newly synthesized protein.