A comparison of the activity, sequence specificity, and CRM1-dependence of different nuclear export signals.
Nuclear export sequences (NESs) have been identified in many cellular proteins, but it remains unclear how different NESs compare in activity. We describe a sensitive new in vivo export assay which we have used to assess the relative export activity of different types of NES. The most common type of export sequence resembles that first identified in the HIV-1 Rev protein and typically comprises a core of large hydrophobic amino acids that specify recognition by the CRM1 export receptor. We compared 10 previously identified Rev-type NESs in our assay, and whereas all were functional, the relative export activities of these signals varied considerably. We further identified 3 new Rev-type NESs from a computer database search, and each export signal was assigned a score of 1 to 9 and ranked in order of activity (e.g., PKI > c-ABL > Ran-BP1 > FMRP > PML > IkappaB-alpha > hdm2). The weakest NESs were found in the p53 tumor suppressor and the p53-regulated proteins p21 and hdm2, which are all normally localized to the nucleus. All of the Rev-type NESs were inactivated by mutation of key hydrophobic residues and by treatment with the CRM1-specific export inhibitor, leptomycin B. In contrast, a different type of export signal, the KNS shuttling element derived from hnRNP K, exhibited a modest export activity that was insensitive to leptomycin B treatment. KNS thus appears to mediate export via a CRM1-independent pathway. Mutagenesis of the KNS sequence identified, for the first time, specific serines and acidic residues necessary for its export activity, thereby distinguishing KNS from other types of nuclear transport signal. We have shown that different nuclear export signals can vary profoundly in activity and therefore conclude that the nuclear export rate of a specific shuttling protein largely depends on both the strength and the accessibility of its NES.
Henderson
B R
BR
Westmead Institute for Cancer Research, University of Sydney, Westmead, New South Wales, 2145, Australia. berich@westgate.wh.usyd
Eleftheriou
A
A
D000595:Amino Acid Sequence; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D001483:Base Sequence; D001943:Breast Neoplasms; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D019992:Databases as Topic; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005260:Female; D015694:Gene Products, rev / Q000737:chemistry* / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins; D028884:Karyopherins; D008164:Luminescent Proteins / Q000378:metabolism; D008545:Melanoma; D008969:Molecular Sequence Data; D000072000:NF-KappaB Inhibitor alpha; D009687:Nuclear Proteins / Q000378:metabolism; D016315:Proto-Oncogene Proteins c-abl / Q000737:chemistry / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013698:Templates, Genetic; D014407:Tumor Cells, Cultured; D054321:rev Gene Products, Human Immunodeficiency Virus; D000097606:Exportin 1 Protein
D000903:Antibiotics, Antineoplastic; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D003609:Dactinomycin; D005231:Fatty Acids, Unsaturated; D015694:Gene Products, rev; D049452:Green Fluorescent Proteins; D020672:I-kappa B Proteins; D028884:Karyopherins; D008164:Luminescent Proteins; D000072000:NF-KappaB Inhibitor alpha; D009687:Nuclear Proteins; D016315:Proto-Oncogene Proteins c-abl; D018160:Receptors, Cytoplasmic and Nuclear; D054321:rev Gene Products, Human Immunodeficiency Virus; D000097606:Exportin 1 Protein; C000606204:NFKBIA protein, human; C038753:leptomycin B