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© The Rockefeller University Press,
0021-9525/1999//673 $5.00
The Journal of Cell Biology, Volume 145, Number 4,
, 1999 673-688
Regular Articles |
Fusogenic Domains of Golgi Membranes Are Sequestered into Specialized Regions of the Stack that Can Be Released by Mechanical Fragmentation
Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
A well-characterized cell-free assay that reconstitutes Golgi transport is shown to require physically fragmented Golgi fractions for maximal activity. A Golgi fraction containing large, highly stacked flattened cisternae associated with coatomer-rich components was inactive in the intra-Golgi transport assay. In contrast, more fragmented hepatic Golgi fractions of lower purity were highly active in this assay. Control experiments ruled out defects in glycosylation, the presence of excess coatomer or inhibitory factors, as well as the lack or consumption of limiting diffusible factors as responsible for the lower activity of intact Golgi fractions. Neither Brefeldin A treatment, preincubation with KCl (that completely removed associated coatomer) or preincubation with imidazole buffers that caused unstacking, activated stacked fractions for transport. Only physical fragmentation promoted recovery of Golgi fractions active for transport in vitro. Rate-zonal centrifugation partially separated smaller transport-active Golgi fragments with a unique v-SNARE pattern, away from the bulk of Golgi-derived elements identified by their morphology and content of Golgi marker enzymes (N-acetyl glucosaminyl and galactosyl transferase activities). These fragments released during activation likely represent intra-Golgi continuities involved in maintaining the dynamic redistribution of resident enzymes during rapid anterograde transport of secretory cargo through the Golgi in vivo.
Key Words: protein transport • Golgi • vesicular transport • secretion
Abbreviations used in this paper: ARF, ADP-ribosylation factor; BFA, Brefeldin A; COP I, coatomer protein; GalT sp. act., galactosyl transferase–specific activity; NAGT, N-acetylglucosamine transferase; VSV-G, envelope glycoprotein of vesicular stomatitis virus; WT, wild-type.
This paper is dedicated to the memory of the late Dr. Wei Lai (see footnote 2). We also thank Scott Berger, as well as Drs. Kathryn Howell, Peggy Weidman, Ben Glick, Jacques Paiement, J. Ostermann, and Charlie Smith for multiple discussions and critical reading of the manuscript; Dr. Wanjin Hong kindly provided affinity-purified antibodies to Golgi SNARE proteins.
Drs. Dominguez and Fazel contributed equally to this work and should be considered co-first authors.
Dr. Dahan's present address is Dept. of Biochemistry & Molecular Biology, Center for Basic Research in Digestive Diseases, Mayo Clinic & Foundation (Gugg. 1701), 200 First St., SW, Rochester, MN 55905. Tel.: (507) 284-0580. Fax: (507) 284-0762. E-mail: dahan.sophie{at}mayo.edu
Dr. Dominguez's present address is Dept. of Biochemistry & Molecular Biology, Thoracic Diseases Research Unit, Mayo Clinic & Foundation (Gugg. 663), 200 First St., SW, Rochester, MN 55905. Tel.: (507) 266-4455. Fax: (507) 284-4452. E-mail: dominguez.michel{at}mayo.edu
2. Dr. Wei Lai (W) first uncovered that Golgi fractions (G) could be isolated from low-speed pellets traditionally referred to as the nuclear (N) pellet; hence the designation WNG fraction.
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