Abstract
The rate-limiting step in the transit of absorbed dietary fat across the enterocyte is the generation of the pre-chylomicron transport vesicle (PCTV) from the endoplasmic reticulum (ER). This vesicle does not require coatomer-II (COPII) proteins for budding from the ER membrane and contains vesicle-associated membrane protein 7, found in intestinal ER, which is a unique intracellular location for this SNARE protein. We wished to identify the protein(s) responsible for budding this vesicle from ER membranes in the absence of the requirement for COPII proteins. We chromatographed rat intestinal cytosol on Sephacryl S-100 and found that PCTV budding activity appeared in the low molecular weight fractions. Additional chromatographic steps produced a single major and several minor bands on SDS-PAGE. By tandem mass spectroscopy, the bands contained both liver and intestinal fatty acid-binding proteins (L- and I-FABP) as well as four other proteins. Recombinant proteins for each of the six proteins identified were tested for PCTV budding activity; only L-FABP and I-FABP (23% the activity of L-FABP) were active. The vesicles generated by L-FABP were sealed, contained apolipoproteins B48 and AIV, were of the same size as PCTV on Sepharose CL-6B, and by electron microscopy, excluded calnexin and calreticulin but did not fuse with cis-Golgi nor did L-FABP generate COPII-dependent vesicles. Gene-disrupted L-FABP mouse cytosol had 60% the activity of wild type mouse cytosol. We conclude that L-FABP can select cargo for and bud PCTV from intestinal ER membranes.
Highlights
The rate-limiting step in the transit of absorbed dietary fat across the enterocyte is the generation of the pre-chylomicron transport vesicle (PCTV) from the endoplasmic reticulum (ER)
In considering potential proteins associated with PCTV budding activity from intestinal ER, we first identified as a paradigm the COPII protein complex (Ϸ576 kDa), which is required for protein vesicle budding
Because we previously found that vesicles generated from intestinal ER using cytosol depleted of Sar1 were incapable of fusing with Golgi [28], we hypothesized that the L-FABPgenerated vesicles, which were formed in a cytosol-free medium, would likely contain low levels of the cytosolic COPII protein, Sar1, on their surface
Summary
The rate-limiting step in the transit of absorbed dietary fat across the enterocyte is the generation of the pre-chylomicron transport vesicle (PCTV) from the endoplasmic reticulum (ER). When 30 mg of native cytosol protein was chromatographed on a Sephacryl S-100 column, the results showed that most of the PCTV-generating activity appeared just before the elution of cytochrome c (Ϸ12 kDa) (Fig. 1A), contrary to our expectations, indicating that either a small molecular weight protein or a complex of very small proteins provided PCTV budding activity.
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