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 t he regulation of a variety of cellular processes including cell. cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nucl ear structure, and DNA synthesis. It is not known whether Ran function s directly in each process or whether many of its roles may be seconda ry 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 effe ctor 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 exc hange stimulated by the Ran-specific guanine nucleotide exchange facto r, RCC1. T42A-Ran.GDP also retains the ability to bind p10/NTF2, a com ponent of the nuclear import pathway. In contrast to wild-type Ran, T4 2A-Ran.GTP binds very weakly or not detectably to three proposed Ran e ffectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP 2, a nucleoporin), and karyopherin beta (a component of the nuclear pr otein 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 digitoni n 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 d efine the exposed region of Ran containing the mutation as a probable effector loop.