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. 1999 Jul 12;146(1):85–98. doi: 10.1083/jcb.146.1.85

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© 1999 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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Polyclonal IgG against Vps33p inhibits cell-free reconstitution of intercompartmental transport between donor and acceptor membranes. (A) Specificity of the anti-Vps33p serum. Whole yeast cells (SEY6210) containing a plasmid with the VPS33 gene under control of the GPD1 promoter (pGPD426-33) were radiolabeled for 10 min (Tran³⁵S-label) and chased (methionine and cysteine) for 30 min. After cell lysis, the extracts were immunoprecipitated with preimmune (P, lane 1) or immune (I, lane 2) serum against Vps33p, subjected to SDS-PAGE, and autoradiography. The positions of molecular weight standards are given in kD. (B) Purified immune IgG inhibits cell-free transport of CPY. Protein A–Sepharose was used to purify IgG from both preimmune and immune rabbit serum. Different amounts (as indicated) of the purified IgG were used in standard cell-free reactions with donor and acceptor membranes. All components of the reactions were added and incubated on ice with the indicated amounts of preimmune and immune IgG for 15 min before the standard 60-min incubation at 25°C. All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography.

Inline graphic — Polyclonal IgG against Vps33p inhibits cell-free reconstitution of intercompartmental transport between donor and acceptor membranes. (A) Specificity of the anti-Vps33p serum. Whole yeast cells (SEY6210) containing a plasmid with the VPS33 gene under control of the GPD1 promoter (pGPD426-33) were radiolabeled for 10 min (Tran³⁵S-label) and chased (methionine and cysteine) for 30 min. After cell lysis, the extracts were immunoprecipitated with preimmune (P, lane 1) or immune (I, lane 2) serum against Vps33p, subjected to SDS-PAGE, and autoradiography. The positions of molecular weight standards are given in kD. (B) Purified immune IgG inhibits cell-free transport of CPY. Protein A–Sepharose was used to purify IgG from both preimmune and immune rabbit serum. Different amounts (as indicated) of the purified IgG were used in standard cell-free reactions with donor and acceptor membranes. All components of the reactions were added and incubated on ice with the indicated amounts of preimmune and immune IgG for 15 min before the standard 60-min incubation at 25°C. All reactions were immunoprecipitated for CPY, subjected to SDS-PAGE, and autoradiography.