Gene replacement reveals that p115/SNARE interactions are essential for Golgi biogenesis.
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.
Puthenveedu
Manojkumar A
MA
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA. linstedt@andrew.cmu.edu
Linstedt
Adam D
AD
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000502:physiology*; D002417:Cattle; D006056:Golgi Apparatus / Q000502:physiology*; D000077614:Golgi Matrix Proteins; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000502:physiology*; D034622:RNA Interference; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; D014759:Viral Envelope Proteins / Q000378:metabolism
D002352:Carrier Proteins; C028391:G protein, vesicular stomatitis virus; D000077614:Golgi Matrix Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; D014759:Viral Envelope Proteins; C091580:vesicular transport factor p115