Role of the Golgi apparatus in the phosphorylation of apolipoprotein B.
Rat hepatic Golgi apparatus-rich fractions were utilized in an in vitro phosphorylation system containing [gamma-32P]ATP to investigate the phosphorylation of apolipoproteins (apo) B48 and B100. Our results demonstrate that the Golgi apparatus contains a kinase(s) that phosphorylates both apoB48 and apoB100 as well as 290- and 460-kDa proteins recognized by antibody to apoB. We refer to the latter proteins as apoB57 and apoB90, respectively. Phosphorylations in the presence of Triton X-100, which increases the permeability of the membranes, or alamethicin, an ionophore that facilitates transmembrane diffusion of ATP, indicate that the active site of the kinase is on the luminal side of the membranes. However, studies with EDTA and EGTA, which are inhibitory to the kinase, suggest binding sites for Mg2+ and perhaps Ca2+ on the cytosolic membrane face. Phosphorylation of apoB was not stimulated by cAMP nor inhibited by protein kinase inhibitor peptide (5-24), indicating that cAMP dependent protein kinase was not involved in the phosphorylation process. Sodium carbonate treatment of the phosphorylated fraction, which permits separation of membrane and luminal contents, revealed that phosphorylated apoB90 and apoB57 are associated primarily with the membrane, whereas phosphorylated apoB48 is found in luminal contents as well as with the membranes. Phosphorylated apoB100 was found primarily with the membrane fraction. No evidence was found for phosphorylation of apoB in rough endoplasmic reticulum fractions. These studies demonstrate the importance of the Golgi apparatus as a subcellular site for the phosphorylation of apoB and suggest that apoB phosphorylation may be important in the assembly and secretion of apoB-containing lipoproteins.
Swift
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Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2561, USA.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000408:Alamethicin / Q000494:pharmacology; D000818:Animals; D053299:Apolipoprotein B-100; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D006056:Golgi Apparatus / Q000378:metabolism*; D008099:Liver / Q000378:metabolism / Q000648:ultrastructure; D017830:Octoxynol / Q000494:pharmacology; D010766:Phosphorylation; D051381:Rats
D053299:Apolipoprotein B-100; D053283:Apolipoprotein B-48; D001055:Apolipoproteins B; D000408:Alamethicin; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D017830:Octoxynol; D004492:Edetic Acid; D000242:Cyclic AMP