Nm23H2 facilitates coat protein complex II assembly and endoplasmic reticulum export in mammalian cells.
The cytosolic coat protein complex II (COPII) mediates vesicle formation from the endoplasmic reticulum (ER) and is essential for ER-to-Golgi trafficking. The minimal machinery for COPII assembly is well established. However, additional factors may regulate the process in mammalian cells. Here, a morphological COPII assembly assay using purified COPII proteins and digitonin-permeabilized cells has been applied to demonstrate a role for a novel component of the COPII assembly pathway. The factor was purified and identified by mass spectrometry as Nm23H2, one of eight isoforms of nucleoside diphosphate kinase in mammalian cells. Importantly, recombinant Nm23H2, as well as a catalytically inactive version, promoted COPII assembly in vitro, suggesting a noncatalytic role for Nm23H2. Consistent with a function for Nm23H2 in ER export, Nm23H2 localized to a reticular network that also stained for the ER marker calnexin. Finally, an in vivo role for Nm23H2 in COPII assembly was confirmed by isoform-specific knockdown of Nm23H2 by using short interfering RNA. Knockdown of Nm23H2, but not its most closely related isoform Nm23H1, resulted in diminished COPII assembly at steady state and reduced kinetics of ER export. These results strongly suggest a previously unappreciated role for Nm23H2 in mammalian ER export.
Kapetanovich
Lori
L
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Baughman
Cassandra
C
Lee
Tina H
TH
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D022181:COP-Coated Vesicles / Q000378:metabolism*; D037281:Calnexin / Q000378:metabolism; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D007700:Kinetics; D013058:Mass Spectrometry; D018613:Microscopy, Confocal; D009701:Nucleoside-Diphosphate Kinase / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000096:biosynthesis / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D015415:Biomarkers; D007527:Isoenzymes; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D037281:Calnexin; D009701:Nucleoside-Diphosphate Kinase; D004072:Digitonin