A nuclear export sequence in GPN-loop GTPase 1, an essential protein for nuclear targeting of RNA polymerase II, is necessary and sufficient for nuclear export.
XAB1/Gpn1 is a GTPase that associates with RNA polymerase II (RNAPII) in a GTP-dependent manner. Although XAB1/Gpn1 is essential for nuclear accumulation of RNAPII, the underlying mechanism is not known. A XAB1/Gpn1-EYFP fluorescent protein, like endogenous XAB1/Gpn1, localized to the cytoplasm but it rapidly accumulated in the cell nucleus in the presence of leptomycin B, a chemical inhibitor of the nuclear transport receptor Crm1. Crm1 recognizes short peptides in substrate proteins called nuclear export sequences (NES). Here, we employed site-directed mutagenesis and fluorescence microscopy to assess the functionality of all six putative NESs in XAB1/Gpn1. Mutating five of the six putative NESs did not alter the cytoplasmic localization of XAB1/Gpn1-EYFP. However, a V302A/L304A double mutant XAB1/Gpn1-EYFP protein was clearly accumulated in the cell nucleus, indicating the disruption of a functional NES. This functional XAB1/Gpn1 NES displays all features present in most common and potent NESs, including, in addition to Φ1-Φ4, a critical fifth hydrophobic amino acid Φ0. Therefore, in human Gpn1 this NES spans amino acids 292-LERLRKDMGSVAL-304. XAB1/Gpn1 NES is remarkably conserved during evolution. XAB1/Gpn1 NES was sufficient for nuclear export activity, as it caused a complete exclusion of EYFP from the cell nucleus. Molecular modeling of XAB1/Gpn1 provided a mechanistic reason for NES selection, as functionality correlated with accessibility, and it also suggested a mechanism for NES inhibition by intramolecular masking. In conclusion, we have identified a highly active, evolutionarily conserved NES in XAB1/Gpn1 that is critical for nucleo-cytoplasmic shuttling and steady-state cytoplasmic localization of XAB1/Gpn1.
Instituto de Física, Universidad Autónoma de San Luis Potosí, Zona Universitaria, San Luis Potosí, San Luis Potosí, México.
Humberto
H
Reyes-Pardo
Angel A
AA
Barbosa-Camacho
Ana E
AE
Pérez-Mejía
Bárbara
B
Lara-Chacón
Leslie A
LA
Salas-Estrada
Angélica Y
AY
Robledo-Rivera
Gabriela M
GM
Montero-Morán
Samuel
S
Lara-González
Mónica R
MR
Calera
Roberto
R
Sánchez-Olea
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000201:enzymology*; D017124:Conserved Sequence / Q000235:genetics; D019143:Evolution, Molecular; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D049790:Nuclear Export Signals; D017433:Protein Structure, Secondary; D012319:RNA Polymerase II / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D049790:Nuclear Export Signals; D011993:Recombinant Fusion Proteins; D012319:RNA Polymerase II; C472669:GPN1 protein, human; D019204:GTP-Binding Proteins