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Sec34p, a Protein Required for Vesicle Tethering to the Yeast Golgi Apparatus, Is in a Complex with Sec35p

Sec34p, a Protein Required for Vesicle Tethering to the Yeast Golgi Apparatus, Is in a Complex... A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34 , a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics . 142:393–406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N -ethylmaleimide sensitive fusion protein attachment protein receptor)–associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1 , a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an ∼750-kD protein complex. Finally, we describe RUD3 , a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1 , which suppresses the sec34-2 mutation as well. Sec34p Sec35p Rud3p vesicle tethering secretory pathway Footnotes Stephanie K. Sapperstein's present address is Department of Neurobiology, Stanford University Medical Center, Stanford, CA 94305. Vladimir V. Lupashin's present address is Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205. Abbreviations used in this paper: CEN, centromere; Gal4p-AD, Gal4p transcriptional activation domain; Gal4p-BD, Gal4p DNA-binding domain; gp-α-factor, glycosylated pro-α-factor; GST, glutathione S-transferase; ORF, open reading frame; PGK, phosphoglycerate kinase; SNARE, soluble N -ethylmaleimide sensitive fusion protein attachment protein receptor; t-SNAREs, SNAREs found predominantly on target membranes; v-SNAREs, SNAREs found predominantly on vesicles. Submitted: 27 July 1999 Revision requested 6 October 1999 Accepted: 12 October 1999 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Cell Biology Rockefeller University Press

Sec34p, a Protein Required for Vesicle Tethering to the Yeast Golgi Apparatus, Is in a Complex with Sec35p

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Publisher
Rockefeller University Press
Copyright
© 1999 The Rockefeller University Press
ISSN
0021-9525
eISSN
1540-8140
DOI
10.1083/jcb.147.4.729
Publisher site
See Article on Publisher Site

Abstract

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34 , a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics . 142:393–406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N -ethylmaleimide sensitive fusion protein attachment protein receptor)–associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1 , a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an ∼750-kD protein complex. Finally, we describe RUD3 , a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1 , which suppresses the sec34-2 mutation as well. Sec34p Sec35p Rud3p vesicle tethering secretory pathway Footnotes Stephanie K. Sapperstein's present address is Department of Neurobiology, Stanford University Medical Center, Stanford, CA 94305. Vladimir V. Lupashin's present address is Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205. Abbreviations used in this paper: CEN, centromere; Gal4p-AD, Gal4p transcriptional activation domain; Gal4p-BD, Gal4p DNA-binding domain; gp-α-factor, glycosylated pro-α-factor; GST, glutathione S-transferase; ORF, open reading frame; PGK, phosphoglycerate kinase; SNARE, soluble N -ethylmaleimide sensitive fusion protein attachment protein receptor; t-SNAREs, SNAREs found predominantly on target membranes; v-SNAREs, SNAREs found predominantly on vesicles. Submitted: 27 July 1999 Revision requested 6 October 1999 Accepted: 12 October 1999

Journal

The Journal of Cell BiologyRockefeller University Press

Published: Nov 15, 1999

References