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 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.
Highlights
Intestinal epithelial cells absorb, process and secrete dietary fat in the form of triacylglycerol-rich chylomicrons
Inhibitors of protein kinase C (PKC) inhibit pre-chylomicron transport vesicle (PCTV) budding ATP is required for the budding of PCTVs from intestinal endoplasmic reticulum (ER) membranes (Siddiqi et al, 2003), which suggests that the budding event is associated with the activity of a protein kinase
We first determined whether protein(s) were phosphorylated under appropriate conditions for PCTV budding from intestinal ER membranes by using [γ-32P]ATP
Summary
Intestinal epithelial cells (enterocytes) absorb, process and secrete dietary fat in the form of triacylglycerol-rich chylomicrons. The rate-limiting step in this multi-event process is the exit of nascent chylomicrons from their site of synthesis, the endoplasmic reticulum (ER) (Mansbach and Dowell, 2000). The nascent chylomicrons leave the ER in a specialized vesicle, the pre-chylomicron transport vesicle (PCTV), that transports them uni-directionally to the cisGolgi (Kumar and Mansbach, 2nd, 1999). There are important differences between PCTVs and vesicles that transport newly synthesized proteins (hereafter referred to as ‘protein vesicles’) from the ER to the Golgi. The large chylomicrons require that PCTVs are 250 nm or more in diameter (Siddiqi et al, 2003). PCTV do not require coatomer protein-II (COPII) proteins for the budding from ER membranes, but COPII proteins are required for the budding of protein vesicles (Siddiqi et al, 2003)
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