A T42A Ran mutation: differential interactions with effectors and regulators, and defect in nuclear protein import.
Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran.GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran.GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin beta (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.
Murphy
G A
GA
Department of Biochemistry, New York University Medical Center, New York, New York 10016, USA.
Moore
M S
MS
Drivas
G
G
Pérez de la Ossa
P
P
Villamarin
A
A
D'Eustachio
P
P
Rush
M G
MG
D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism / Q000494:pharmacology; D018797:Cell Cycle Proteins; D002463:Cell Membrane Permeability / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology; D020690:GTPase-Activating Proteins / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018832:Molecular Chaperones; D009154:Mutation / Q000235:genetics; D009685:Nuclear Envelope / Q000187:drug effects / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D029543:Nucleocytoplasmic Transport Proteins; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D028961:beta Karyopherins; D020931:ran GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D018832:Molecular Chaperones; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; C503612:Nutf2 protein, rat; D011993:Recombinant Fusion Proteins; D028961:beta Karyopherins; C091775:ran-binding protein 1; C094493:ran-binding protein 2; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020931:ran GTP-Binding Protein; D004072:Digitonin