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Published July 15, 1991 | Published
Journal Article Open

Compartmental organization of Golgi-specific protein modification and vacuolar protein sorting events defined in a yeast sec18 (NSF) mutant


The sec18 and sec23 secretory mutants of Saccharomyces cerevisiae have previously been shown to exhibit temperature-conditional defects in protein transport from the ER to the Golgi complex (Novick, P., S. Ferro, and R. Schekman, 1981. Cell. 25:461-469). We have found that the Sec18 and Sec23 protein functions are rapidly inactivated upon shifting mutant cells to the nonpermissive temperature (less than 1 min). This has permitted an analysis of the potential role these SEC gene products play in transport events distal to the ER. The sec-dependent transport of alpha-factor (alpha f) and carboxypeptidase Y (CPY) biosynthetic intermediates present throughout the secretory pathway was monitored in temperature shift experiments. We found that Sec18p/NSF function was required sequentially for protein transport from the ER to the Golgi complex, through multiple Golgi compartments and from the Golgi complex to the cell surface. In contrast, Sec23p function was required in the Golgi complex, but only for transport of alpha f out of an early compartment. Together, these studies define at least three functionally distinct Golgi compartments in yeast. From cis to trans these compartments contain: (a) An alpha 1→6 mannosyltransferase; (b) an alpha 1→3 mannosyltransferase; and (c) the Kex2 endopeptidase. Surprisingly, we also found that a pool of Golgi-modified CPY (p2 CPY) located in a compartment distal to the alpha 1→3 mannosyltransferase does not require Sec18p function for final delivery to the vacuole. This compartment appears to be equivalent to the Kex2 compartment as we show that a novel vacuolar CPY-alpha f-invertase fusion protein undergoes efficient Kex2-dependent cleavage resulting in the secretion of invertase. We propose that this Kex2 compartment is the site in which vacuolar proteins are sorted from proteins destined to be secreted.

Additional Information

© 1991 by The Rockefeller University Press Received for publication 24 January 1991 and in revised form 11 March 1991. We would like to thank Randy Schekman for supplying antibodies against of and specific carbohydrate linkages. We are particularly grateful to Bruce Horazdovsky, Jane Robinson, Jeff Stack, Paul Herman, Tom Vida, Gerhard Paravicini, and Karl Köhrer for helpful discussion and critically reading this manuscript, and also to Nancy King for secretarial assistance. This work was supported by Public Health Service grant GM-32703 from the National Institutes of Health to S.D. Emr. T.R. Graham was supported by a postdoctoral research fellowship from the American Cancer Society.

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