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J. Cell Biol.,
Volume 145, Number 4, May 17, 1999 673-688
* Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada, H3A 2B2; and 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.
Department of
Cell Biology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7
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