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© The Rockefeller University Press, 0021-9525/1999/12/1457/ $5.00
The Journal of Cell Biology, Volume 147, Number 7, December 27, 1999 1457-1472

Original Article

ER/Golgi Intermediates Acquire Golgi Enzymes by Brefeldin A–Sensitive Retrograde Transport In Vitro

Chung-Chih Lina, Harold D. Lovea, Jennifer N. Gushueb, John J.M. Bergeronb, and Joachim Ostermanna
a Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
b Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, H3A 2B2 Canada

Correspondence to: Joachim Ostermann, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146. Tel:(615) 343-3803 Fax:(615) 343-0704 E-mail:ostermj{at}ctrvax.vanderbilt.edu.

Secretory proteins exit the ER in transport vesicles that fuse to form vesicular tubular clusters (VTCs) which move along microtubule tracks to the Golgi apparatus. Using the well-characterized in vitro approach to study the properties of Golgi membranes, we determined whether the Golgi enzyme NAGT I is transported to ER/Golgi intermediates. Secretory cargo was arrested at distinct steps of the secretory pathway of a glycosylation mutant cell line, and in vitro complementation of the glycosylation defect was determined. Complementation yield increased after ER exit of secretory cargo and was optimal when transport was blocked at an ER/Golgi intermediate step. The rapid drop of the complementation yield as secretory cargo progresses into the stack suggests that Golgi enzymes are preferentially targeted to ER/Golgi intermediates and not to membranes of the Golgi stack. Two mechanisms for in vitro complementation could be distinguished due to their different sensitivities to brefeldin A (BFA). Transport occurred either by direct fusion of preexisting transport intermediates with ER/Golgi intermediates, or it occurred as a BFA-sensitive and most likely COP I–mediated step. Direct fusion of ER/Golgi intermediates with cisternal membranes of the Golgi stack was not observed under these conditions.

Key Words: Golgi apparatus, in vitro transport, secretion, transport vesicles, ER


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