IN-VITRO AND IN-VIVO EVIDENCE THAT PROTEIN AND U1 SNRNP NUCLEAR IMPORT IN SOMATIC-CELLS DIFFER IN THEIR REQUIREMENT FOR GTP-HYDROLYSIS, RANTC4 AND RCC1/

GTP-hydrolysis, the small ras-related GTP-binding protein Ran and its cognate guanosine nucleotide exchange factor, the RCC1 gene product, h ave recently been identified as essential components of the protein nu clear import pathway, In this report we use three independent approach es to investigate the role of these components in U1 snRNP nuclear imp ort in somatic cells, (i) Using a somatic cell based in vitro nuclear import system we show that U1 snRNP nuclear import, in marked contrast to protein transport, is not significantly inhibited by non-hydrolyza ble GTP-analogs and is therefore unlikely to require GTP-hydrolysis. ( ii) Using the dominant negative Ran mutant RanQ69L, which is defective in GTP-hydrolysis, we show that Ran-mediated GTP-hydrolysis is not es sential for the nuclear import of U1 snRNP in microinjected cultured c ells, (iii) Using a cell line expressing a thermolabile RCC1 gene prod uct, we show that the nuclear accumulation of microinjected U1 snRNP i s not significantly affected by RCC1 depletion at the non-permissive t emperature, indicating that RCC1 function is not essential for U-snRNP nuclear import, Based on these observations we conclude that protein and U-snRNP nuclear import in somatic cells differ in their requiremen ts for GTP-hydrolysis, and Ran or RCC1 function, Based on these result s, the substrates for nucleocytoplasmic exchange across the NPC can be divided into two classes, those absolutely requiring Ran, including p rotein import and mRNA export, and those for which Ran is not essentia l, including U-snRNP nuclear import, together with tRNA and U1 snRNA n uclear export.