Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 101, Issue 5, Pages 1253-1256Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0306373101
Keywords
Golgi apparatus; RNA interference; giantin; GM130
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Funding
- NIGMS NIH HHS [GM-56779-02, R01 GM056779] Funding Source: Medline
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Functional characterization of protein interactions in mammalian systems has been hindered by the inability to perform complementation analyses in vivo. Here,we use functional replacement of the vesicle docking protein p115 to separate its essential from its nonessential interactions. p115 is required for biogenesis of the Golgi apparatus, but it is unclear whether its mechanism of action requires its golgin and/or SNARE interactions. Short interfering RNA-mediated knockdown of p115 induced extensive Golgi fragmentation and impaired secretory traffic. Reassembly of a structurally and functionally normal Golgi occurred on expression of a p115 homologue not recognized by the short interfering RNA. Strikingly, versions of p115 lacking its phosphorylation site and the golgin-binding domains also restored the Golgi apparatus in cells lacking endogenous p115. In contrast, the p115 SNARE-interacting domain was required for Golgi biogenesis. This suggests that p115 acts directly, rather than via a tether, to catalyze trans-SNARE complex formation preceding membrane fusion.
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