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First published online 4 December 2002
doi: 10.1242/jcs.00215
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Research Article |
1 Division of Gastroenterology, University of Tennessee Health Science Center,
Memphis, TN 38163, USA
2 Veterans Affairs Medical Center, Memphis, TN 38104, USA
3 VA Connecticut Healthcare, West Haven, CT 06516, USA
4 Department of Medicine, Yale University School of Medicine, New Haven, CT
06520, USA
* Author for correspondence (e-mail: cmansbach{at}utmem.edu)
Accepted 10 October 2002
The budding of vesicles from endoplasmic reticulum (ER) that contains nascent proteins is regulated by COPII proteins. The mechanisms that regulate lipid-carrying pre-chylomicron transport vesicles (PCTVs) budding from the ER are unknown. To study the dependence of PCTV-ER budding on COPII proteins we examined protein and PCTV budding by using ER prepared from rat small intestinal mucosal cells prelabeled with 3H-oleate or 14C-oleate and 3H-leucine. Budded 3H-oleate-containing PCTVs were separated by sucrose density centrifugation and were revealed by electron microscopy as 142-500 nm vesicles. Our results showed the following: (1) Proteinase K treatment did not degrade the PCTV cargo protein, apolipoprotein B-48, unless Triton X-100 was added. (2) PCTV budding was dependent on cytosol and ATP. (3) The COPII proteins Sar1, Sec24 and Sec13/31 and the membrane proteins syntaxin 5 and rBet1 were associated with PCTVs. (4) Isolated PCTVs were able to fuse with intestinal Golgi. (5) Antibodies to Sar1 completely inhibited protein vesicle budding but increased the generation of PCTV; these changes were reversed by the addition of recombinant Sar1. (6) PCTVs formed in the absence of Sar1 did not contain the COPII proteins Sar1, Sec24 or Sec31 and did not fuse with the Golgi complex. Together, these findings suggest that COPII proteins may not be required for the exit of membrane-bound chylomicrons from the ER but that they or other proteins may be necessary for PCTV fusion with the Golgi.
Key words: Pre-chylomicron transport vesicle, PCTV, COPII proteins, Vesicle budding, Lipid absorption, Sar1, Chylomicrons
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