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First published online February 22, 2006
doi: 10.1242/10.1242/jcs.02803

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Vesicle-associated membrane protein 7 is expressed in intestinal ER

¹ Division of Gastroenterology, The University of Tennessee Health Science Center, Memphis, TN 38163 USA
² Veterans Affairs Medical Center, Memphis, TN 38163 USA
³ Department of Medicine, VA Healthcare, and Yale University School of Medicine, New Haven, CT 06516 USA

View larger version (26K): [in a new window]   Fig. 1. Immunoblots demonstrating that VAMP7 is found in enterocytes and concentrated on PCTVs. (A) Immunoblot of rat intestinal ER (30 µg protein) separated by SDS-PAGE, transblotted to nitrocellulose membrane, and probed with anti-VAMP7 antibody. The molecular sizes of identified bands are indicated. (B) Immunoblot of ER membrane separated by 2D SDS-PAGE (8-18%). ER membranes (200 µg) were solubilized and separated first by pI and subsequently by molecular size (Materials and Methods). The proteins were transblotted onto nitrocellulose membranes and probed with anti-VAMP7 antibodies. Detection was by ECL. (C) Immunoblots of VAMP7 in different rat organs. of Post nuclear supernatant (30 µg protein) of rat intestinal (Inst.), rat kidney (Kid.), and rat liver (Liv.) were separated by 12% SDS-PAGE, transblotted onto nitrocellulose membranes and immunoblotted for VAMP7. Bands at 25 and 18 kDa are indicated.

View larger version (31K): [in a new window]   Fig. 2. Immunoblots of VAMP7, GOS28, apoB48, apoAIV, Ykt6, syntaxin8 and Sec22b in subcellular fractions of rat intestinal cells. Whole-cell lysate, PCTV, ER and Golgi proteins (30 µg protein) were separated by 12% SDS-PAGE, transblotted onto nitrocellulose membranes and immunoblotted for VAMP7, Sec22b, GOS28, apoB48, apoAIV, Ykt6 and Syntaxin8.

View larger version (70K): [in a new window]   Fig. 3. Deconvoluted microscopy of isolated rat intestinal cells showing colocalization of VAMP7 with the ER marker, PDI. Optical horizontal sections stained with anti-VAMP7 antibodies (Texas Red), anti-PDI antibody (FITC green) and the merged image. Arrows indicate structures resembling ER. Bars, 5 µm.

View larger version (125K): [in a new window]   Fig. 4. Immunogold labeling of PCTV showing VAMP7 and either Sar1 or rBet1 to be present on the same membrane as shown by electron microscopy. PCTVs were placed on formvar-coated Nickel grids and exposed to pre-immune rabbit IgG (A), anti-Sar1 and anti-VAMP7 (B) or anti-rBet1 and anti-VAMP7 antibodies (C). Rabbit anti-VAMP7 antibodies were labeled with 15 nm immunogold particles. Rabbit anti-Sar1 and mouse anti-rBet1 antibodies were labeled with 10 nm immunogold particles. Bars, 100 µm (A); 80 µm (B,C).

View larger version (29K): [in a new window]   Fig. 5. Distribution of rat intestinal subcellular organelles separated on an iodixanol gradient identifying VAMP7 in both ER and endosomes. Post nuclear supernatant protein (2 mg) was separated on a 10-40% iodixanol gradient. The gradient was centrifuged and resolved as described in the Materials and Methods and the fractions obtained indicated. The proteins in each fraction (50 µl) were separated by 12% SDS-PAGE, transblotted to a membrane and the location of calreticulin, VAMP7, Rab11, Sec22b and Syntaxin8 identified by immunoblotting with their specific antibodies.

View larger version (22K): [in a new window]   Fig. 6. Distribution of VAMP7 in the postnuclear supernatants of rat intestine, liver and kidney. Post-nuclear supernatant (2 mg protein) of intestine (A), liver (B) and kidney (C) were separated by 10-40% iodixanol gradient (Materials and Methods) and the gradient resolved by aspiration. Fractions (50 µl) were separated by 12% SDS-PAGE, transblotted to nitrocellulose membranes, and VAMP7 was identified by immunoblotting with anti-VAMP7 antibodies.

View larger version (44K): [in a new window]   Fig. 7. Anti-VAMP7 antibodies inhibit the transfer of TAG but only modestly reduce the transfer of newly synthesized proteins from the ER to the cis Golgi. 3H-TAG-loaded rat intestinal ER (300 µg protein) were incubated with 10 µl pre-immune IgG (A), ER treated with 10 µl of anti-VAMP7 antibody (B), cis Golgi treated with 10 µl anti-VAMP7 antibodies (C), ER treated with anti-syntaxin8 antibodies (D) or ER treated with anti-Sec22b antibodies (E). Unbound antibody was removed by washing and the ER was incubated with 1 mg native rat cytosol, 500 µg rat cis Golgi, an ATP-generating system and buffer B for 30 minutes at 35°C. The cis Golgi was isolated on a sucrose step gradient (see Materials and Methods) and obtained by aspiration. TAG was extracted and the dpm determined. The data are the mean ± s.e.m., n=4. There was a significant difference in 3H-TAG transfer (P<0.01) between VAMP7 antibody-treated ER and the pre-immune control. (B) ER (500 µg protein) loaded with 14C-TAG and 3H-protein was treated with pre-immune IgG (10 µl) (A and C), or anti-VAMP7 antibody (10 µl) (B and D), excess antibody was removed by washing, and incubated with native rat intestinal cytosol (1 mg protein), an ATP-generating system and cis Golgi (500 µg protein). The Golgi fraction was isolated on a sucrose gradient and the amount of TAG (left y axis) or protein (right y axis) dpm determined (Materials and Methods). The percentage reduction in transport upon VAMP7 antibody treatment is shown above the bars. A compared with B, P<0.001; C compared with D, P>0.05. The data are the mean ± s.e.m. (n=4).

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