PKC zeta-mediated phosphorylation controls budding of the pre-chylomicron transport vesicle.
Dietary triacylglycerols are absorbed by enterocytes and packaged in the endoplasmic reticulum (ER) in the intestinal specific lipoprotein, the chylomicron, for export into mesenteric lymph. Chylomicrons exit the ER in an ER-to-Golgi transport vesicle, the pre-chylomicron transport vesicle (PCTV), which is the rate-limiting step in the transit of chylomicrons across the cell. Here, we focus on potential mechanisms of control of the PCTV-budding step from the intestinal ER. We incubated intestinal ER with intestinal cytosol and ATP to cause PCTV budding. The budding reaction was inhibited by 60 nM of the PKC inhibitor Gö 6983, suggesting the importance of PKCzeta in the generation of PCTV. Immunodepletion of PKCzeta from the cytosol and the use of washed ER greatly inhibited the generation of PCTVs, but was restored following the addition of recombinant PKCzeta. Intestinal ER incubated with intestinal cytosol and [gamma-(32)P]ATP under conditions supporting the generation of PCTVs showed the phosphorylation of a 9-kDa band following autoradiography. The phosphorylation of this protein correlated with the generation of PCTVs but not the formation of protein vesicles and was inhibited by depletion of PKCzeta. Phosphorylation of the 9-kDa protein was restored following the addition of recombinant PKCzeta. The association of the 9-kDa protein with proteins that are important for PCTV budding was phosphorylation dependent. We conclude that PKCzeta activity is required for PCTV budding from intestinal ER, and is associated with phosphorylation of a 9-kDa protein that might regulate PCTV budding.
Siddiqi
Shadab A
SA
The Division of Gastroenterology, The University of Tennessee Health Science Center, Memphis, TN 38163, USA.
Mansbach
Charles M
CM
D000818:Animals; D053283:Apolipoprotein B-48 / Q000378:metabolism; D002914:Chylomicrons / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology; D020536:Enzyme Activators / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects; D050556:Fatty Acid-Binding Proteins / Q000378:metabolism; D007422:Intestines / Q000166:cytology / Q000201:enzymology; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D050683:R-SNARE Proteins / Q000378:metabolism; D051381:Rats; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology; D022161:Transport Vesicles / Q000187:drug effects / Q000201:enzymology*; D000099221:Protein Kinase C zeta
D053283:Apolipoprotein B-48; D002914:Chylomicrons; D020536:Enzyme Activators; D050556:Fatty Acid-Binding Proteins; D010750:Phosphoproteins; D011493:Protein Kinase C; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D050683:R-SNARE Proteins; D000099221:Protein Kinase C zeta; C496926:Vamp7 protein, rat