In vivo analysis of the lumenal binding protein (BiP) reveals multiple functions of its ATPase domain.
The endoplasmic reticulum (ER) chaperone binding protein (BiP) binds exposed hydrophobic regions of misfolded proteins. Cycles of ATP hydrolysis and nucleotide exchange on the ATPase domain were shown to regulate the function of the ligand-binding domain in vitro. Here we show that ATPase mutants of BiP with defective ATP-hydrolysis (T46G) or ATP-binding (G235D) caused permanent association with a model ligand, but also interfered with the production of secretory, but not cytosolic, proteins in vivo. Furthermore, the negative effect of BiP(T46G) on secretory protein synthesis was rescued by increased levels of wild-type BiP, whereas the G235D mutation was dominant. Unexpectedly, expression of a mutant BiP with impaired ligand binding also interfered with secretory protein production. Although mutant BiP lacking its ATPase domain had no detrimental effect on ER function, expression of an isolated ATPase domain interfered with secretory protein synthesis. Interestingly, the inhibitory effect of the isolated ATPase was alleviated by the T46G mutation and aggravated by the G235D mutation. We propose that in addition to its role in ligand release, the ATPase domain can interact with other components of the protein translocation and folding machinery to influence secretory protein synthesis.
Snowden
Christopher James
CJ
Centre for Plant Sciences, Faculty of Biological Sciences, The University of Leeds, Leeds LS2 9JT, UK.
Leborgne-Castel
Nathalie
N
Wootton
Lucie Jan
LJ
Hadlington
Jane Louise
JL
Denecke
Jurgen
J
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D006868:Hydrolysis; D047468:Immunoprecipitation; D008954:Models, Biological; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D009154:Mutation; D030821:Plants, Genetically Modified; D011485:Protein Binding; D011523:Protoplasts / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases