Assembly and secretion of very low density lipoproteins containing apolipoprotein B48 in transfected McA-RH7777 cells. Lack of evidence that palmitoylation of apolipoprotein B48 is required for lipoprotein secretion.
We examined the role of S-linked palmitoylation of human apolipoprotein (apo) B in the assembly and secretion of very low density lipoproteins using recombinant human apoB48. There are four free cysteine residues (Cys(1085), Cys(1396), Cys(1478), and Cys(1635)) within apoB48 that potentially can be palmitoylated. All four cysteine residues were substituted with serine by site-specific mutagenesis. The mutant protein was expressed in transfected rat hepatoma McA-RH7777 cells. Metabolic labeling of the stably transfected cells with iodopalmitic acid analog showed that the mutant apoB48 lacked palmitoylation. The lack of palmitoylation had little impact on the ability of apoB48 to assemble and secrete very low density lipoproteins or high density lipoproteins. Immunocytochemistry experiments using confocal microscopy failed to reveal any major alterations in the intracellular distribution of the mutant apoB48 at steady state. Pulse-chase analysis combined with subcellular fractionation showed no apparent deficiency in the movement of the mutant apoB48 protein from the endoplasmic reticulum to cis/medial Golgi. However, the mutant apoB48 lacking palmitoylation showed retarded movement toward the distal Golgi and increased association (>2-fold) with the membranes of the secretory compartments. A marginal decrease (by 15-20%) in secretion efficiency as compared with that of wild type apoB48 was also observed. These results suggest that lack of palmitoylation may influence the partitioning of apoB48 between microsomal membranes and microsomal lumen, but it does not compromise the ability of apoB48 to assemble lipoproteins.
Vukmirica
Jelena
J
Lipoprotein and Atherosclerosis Group, University of Ottawa Heart Institute, Canada K1Y 4W7.
Tran
Khai
K
Liang
Xiquan
X
Shan
Jing
J
Yuan
Jane
J
Miskie
Brooke A
BA
Hegele
Robert A
RA
Resh
Marilyn D
MD
Yao
Zemin
Z
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008074:Lipoproteins / Q000378:metabolism; D008079:Lipoproteins, VLDL / Q000737:chemistry* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010168:Palmitates / Q000494:pharmacology; D019308:Palmitic Acid / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D014461:Ultracentrifugation
D000911:Antibodies, Monoclonal; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B; D008074:Lipoproteins; D008079:Lipoproteins, VLDL; D010168:Palmitates; C022776:2-bromopalmitate; D019308:Palmitic Acid; D012694:Serine; D003545:Cysteine