In vitro and in vivo evidence that protein and U1 snRNP nuclear import in somatic cells differ in their requirement for GTP-hydrolysis, Ran/TC4 and RCC1.
GTP-hydrolysis, the small ras-related GTP-binding protein Ran and its cognate guanosine nucleotide exchange factor, the RCC1 gene product, have recently been identified as essential components of the protein nuclear import pathway. In this report we use three independent approaches to investigate the role of these components in U1 snRNP nuclear import 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-hydrolyzable 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 essential for the nuclear import of U1 snRNP in microinjected cultured cells. (iii) Using a cell line expressing a thermolabile RCC1 gene product, we show that the nuclear accumulation of microinjected U1 snRNP is not significantly affected by RCC1 depletion at the non-permissive temperature, 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 requirements for GTP-hydrolysis, and Ran or RCC1 function. Based on these results, the substrates for nucleocytoplasmic exchange across the NPC can be divided into two classes, those absolutely requiring Ran, including protein import and mRNA export, and those for which Ran is not essential, including U-snRNP nuclear import, together with tRNA and U1 snRNA nuclear export.
Marshallsay
C
C
Institute for Molecular Biology and Tumour Research, Philipps University, Marburg, Germany.
Dickmanns
A
A
Bischoff
F R
FR
Ponstingl
H
H
Fanning
E
E
Lührmann
R
R
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D001692:Biological Transport / Q000502:physiology; D018797:Cell Cycle Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000502:physiology; D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000502:physiology*; D011506:Proteins / Q000378:metabolism*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism*; D020931:ran GTP-Binding Protein
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D011506:Proteins; C062521:RCC1 protein, human; D017412:Ribonucleoprotein, U1 Small Nuclear; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein