Nuclear import and export functions in the different isoforms of the AUF1/heterogeneous nuclear ribonucleoprotein protein family.
The heterogeneous nuclear ribonucleoprotein D family of proteins also known as AUF1 consists of four isoforms implicated in both nuclear and cytoplasmic functions. The AUF1 proteins are largely nuclear but also are found in the cytoplasm and are thought to undergo nucleocytoplasmic shuttling. The nucleocytoplasmic distribution and potential shuttling activity of the individual AUF1 isoforms have not been previously studied in detail. Therefore, we characterized the nucleocytoplasmic transport of each of the heterogeneous nuclear ribonucleoprotein D/AUF1 isoforms. All four AUF1 proteins were found to undergo rapid nucleocytoplasmic shuttling in a manner that is transcription-independent, carrier-mediated, and energy-requiring. Nucleocytoplasmic shuttling of the AUF1 proteins is shown to utilize a novel arrangement of nuclear import and export signals. Mutagenesis of the AUF1 proteins and fusion of polypeptides to a green fluorescent protein reporter demonstrated that a nuclear import signal is located in the C-terminal domain of the protein and is found only in the two smaller isoforms. Further mapping demonstrated that nuclear export is facilitated by sequences in AUF1 exon 7 found in the C-terminal domain of the two larger AUF1 isoforms. A subset of AUF1 proteins are shown to directly interact in vitro using purified recombinant proteins and in vivo in the absence of RNA. These results suggest that nuclear import of AUF1 is facilitated by sequences found only in the two smaller isoforms and that nuclear export is facilitated by sequences (exon 7 and the C-terminal domain) found only in the two larger isoforms. This novel arrangement of signals might represent a mechanism to assure co-shuttling of a subset of AUF1 proteins that interact in a heterocomplex.
Sarkar
Bedabrata
B
Department of Microbiology, New York University School of Medicine, New York, New York 10016, USA.
Lu
Jin-Yu
JY
Schneider
Robert J
RJ
D016475:3T3 Cells; D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D005091:Exons; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007202:Indicators and Reagents / Q000378:metabolism; D007536:Isomerism; D028884:Karyopherins / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D011817:Rabbits; D018160:Receptors, Cytoplasmic and Nuclear; D014158:Transcription, Genetic / Q000502:physiology; D000097606:Exportin 1 Protein
D049452:Green Fluorescent Proteins; D000081035:Heterogeneous Nuclear Ribonucleoprotein D0; D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D007202:Indicators and Reagents; D028884:Karyopherins; D008164:Luminescent Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D000097606:Exportin 1 Protein; C000630986:HNRNPD protein, human; C000630987:Hnrpd protein, mouse