PKC
-mediated phosphorylation controls budding of the pre-chylomicron transport vesicle
Shadab A. Siddiqi1, and
Charles M. Mansbach, II1,2,*
1 The Division of Gastroenterology, The University of Tennessee Health Science Center, 920 Madison Avenue, Memphis, TN 38163, USA
2 Veterans Affairs Medical Center, Memphis, TN, USA
* Author for correspondence (e-mail: cmansbach{at}utmem.edu)
Accepted for publication 22 April 2008.
Abstract:
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 PKC
in the generation of PCTV. Immunodepletion of PKC from the cytosol and the use of washed ER greatly inhibited the generation of PCTVs, but was restored following the addition of recombinant PKC. Intestinal ER incubated with intestinal cytosol and [
-32P]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 PKC. Phosphorylation of the 9-kDa protein was restored following the addition of recombinant PKC. The association of the 9-kDa protein with proteins that are important for PCTV budding was phosphorylation dependent. We conclude that PKC activity is required for PCTV budding from intestinal ER, and is associated with phosphorylation of a 9-kDa protein that might regulate PCTV budding.
Key Words: PKC • Pre-chylomicron transport vesicle • Protein phosphorylation • Chylomicron
