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. 1999 Dec 13;147(6):1223–1236. doi: 10.1083/jcb.147.6.1223

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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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The drs2Δ mutant accumulates aberrant Golgi membranes and exhibits a deficiency of clathrin-coated vesicles. (A) Wild-type and drs2Δ cells were grown at 30°C and shifted to 15°C for 1 h, and then lysed and subjected to centrifugation at 21,000 g for 30 min. The 21,000-g supernatant was centrifuged at 100,000 g for 80 min to generate a 100,000-g pellet. This pellet was resuspended and further resolved on a Sephacryl S-1000 column. Samples of every other fraction from fraction 15 to 35 were assayed by immunoblotting for the clathrin heavy chain (Chc1p), a late Golgi protein Mnn1p, and an endosomal t-SNARE Pep12p. (B–D) Membranes in fraction 16 from the drs2Δ sample where Mnn1p was found (B), and fraction 26 from wild-type (C) and drs2Δ (D) samples where Chc1p was enriched were fixed, pelleted, and examined by EM. Bars, 50 nm.