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 the 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 carboxy-terminal mutated (F388-->Stop, K428-->Stop and K441-->Stop) hLPL cDNAs that were expressed in COS1 cells. Immunoblotting of cell extracts showed that all three constructs led to similar levels of protein. Both wild type (WT) hLPL and the truncated K441-->Stop hLPL were secreted to the extracellular medium, and presented a similar intracellular distribution pattern as shown by immunofluorescence. Neither F388-->Stop nor K428-->Stop hLPL protein was detected in cell medium. Immunofluorescence experiments showed that both truncated hLPL were retained within an intracellular compartment, which became larger. Double immunofluorescence analysis using antibodies against LPL and antiprotein disulfide isomerase as a marker showed that the truncated K428-->Stop hLPL was retained within the rough endoplasmic reticulum. This truncated protein was not found in other compartments in the secretory pathway, such as Golgi complex and lysosomes, indicating that it did not exit the endoplasmic reticulum. Further analysis of the C-terminal region of the LPL molecular model showed both that F388-->Stop and K428-->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, we conclude that the C-terminal domain of hLPL is critical for correct intracellular processing of the newly synthesized protein.
Buscà
R
R
Department of Cell Biology, University of Barcelona, Spain.
Martínez
M
M
Vilella
E
E
Peinado
J
J
Gelpi
J L
JL
Deeb
S
S
Auwerx
J
J
Reina
M
M
Vilaró
S
S
D004252:DNA Mutational Analysis; D018870:Endoplasmic Reticulum, Rough / Q000378:metabolism*; D006801:Humans; D008071:Lipoprotein Lipase / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D008071:Lipoprotein Lipase