Sec24C is required for docking the prechylomicron transport vesicle with the Golgi

Abstract

Journal of Lipid Research Volume 51, 2010 1093anism by which dietary fat is delivered to peripheral targeted tissues, muscle, heart, and adipose tissue. The majority of the triacylglycerol (TAG) in the chylomicron is derived from hydrolytic products of dietary lipids that are resynthesized to TAG by the intestinal ER. The newly synthesized TAG crosses the ER membrane and forms the prechylomicron in the ER lumen in a two-step process ( 3 ). The exit step of the prechylomicron from the ER is the rate-limiting step by which dietary TAG traverses the intestinal absorptive cell ( 4 ) ( 5 ). Once detached from the ER membrane, the PCTV dock and then fuse with the Golgi using the N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex composed of VAMP7 (the R-SNARE), syntaxin5 (the Qa-SNARE), vti1A (the Qb-SNARE), and Bet1 (the Qc-SNARE) ( 2 ). Our interest in the molecular mechanism of the docking of PCTV with the Golgi in relation to the COPII proteins was raised by these fi ndings: (a) Although PCTV could be generated from ER membranes in the absence of Sar1, the initiator of the COPII complex, the vesicles formed could not fuse with the Golgi ( 1 ). The vesicles were of the same size as PCTV generated using native cytosol, contained apolipoproteinB48 (apoB48), the quintessential chylomicron apolipoprotein, and VAMP7, the R-SNARE of the PCTV-Golgi SNARE complex; however, neither docking nor fusion was possible using these vesicles. b) The liver-fatty acid binding protein (L-FABP) can, in the absence of additional cytosolic components, elaborate PCTV from intestinal ER membranes ( 6 ). However, in support of the data obtained using vesicles formed in the absence of Sar1, these L-FABP formed vesicles do not contain COPII proteins and cannot fuse with the Golgi. In Abstract The rate-limiting step in the transit of dietary fat across the intestinal absorptive cell is its exit from the endoplasmic reticulum (ER) in a specialized ER-to-Golgi transport vesicle, the prechylomicron transport vesicle (PCTV). PCTV bud off from the ER membranes and have unique features; they are the largest ER-derived vesicles (average diameter 250 nm), do not require GTP and COPII proteins for their formation, and utilize VAMP7 as a v-N-ethylmaleimide sensitive factor attachment protein receptor (SNARE). However, PCTV require COPII proteins for their fusion with the Golgi, suggesting a role for them in Golgi target recognition. In support of this, PCTV contained each of the fi ve COPII proteins when docked with the Golgi. When PCTV were fused with the Golgi, the COPII proteins were present in greatly diminished amounts, indicating they had cycled back to the cytosol. Immuno-depletion of Sec31 from the cytosol did not affect PCTV-Golgi docking, but depletion of Sec23 resulted in a 25% decrease. Immunodepletion of Sec24C caused a nearly complete cessation of PCTV docking activity, but on the addition of recombinant Sec24C, docking activity was restored. We conclude that the COPII proteins are present at docking of PCTV with...

CideB Protein Is Required for the Biogenesis of Very Low Density Lipoprotein (VLDL) Transport Vesicle

“…As can be seen in Fig. 5C, the Sar1-and Sec24-depleted system did not support the formation of the VTV, which is consistent with published studies (56,57). Taken together, these data strongly suggest that CideB plays an important role in the biogenesis of the VTV.…”

“…Because both Sar1 and Sec24 interact with CideB and apoB100, it is possible that this interaction triggers the process of VTV formation. To test this speculation, we decided to immunodeplete hepatic cytosol with specific antibodies against Sar1 and Sec24 as we have done successfully previously (57). Also, we washed our ER membranes with urea to remove peripheral Sar1 and Sec24 as we have done in the past (21).…”

A novel multiprotein complex is required to generate the prechylomicron transport vesicle from intestinal ER

“…The lack of COPII proteins in these systems is associated with vesicles that may either bypass the Golgi all together, indicating a loss of a Golgi targeting signal ( 36 ), or the vesicles may be targeted to the Golgi despite a lack of the COPII proteins ( 35,37 ). In the case of PCTV, the COPII proteins are required for PCTV-Golgi fusion ( 6,12 ).…”

“…L-FABP initiates PCTV budding ( 13 ). Sec24C, while not required for budding, is required for PCTV docking and fusion with the Golgi ( 12 ). Further, Sec24 has been shown to function in active PCTV budding and consequent escape of PCTV component proteins ( 6 ).…”

Insights from human congenital disorders of intestinal lipid metabolism

“…Although Sar1a antagonizes the lipoprotein secretion-promoting activity of Sar1b, both isoforms were noted to modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo ( 221 ). It is noteworthy that Sar1b GTPase is not only central for the COPII responsible for the biosynthetic transport of proteins from the ER to the Golgi apparatus, but also for fusion of the specifi c CM transport vesicle, the preCM transport vesicle, with the Golgi ( 50, 54,222,223 ).…”