title: The Dbp5 cycle at the nuclear pore complex during mRNA export I: dbp5 mutants with defects in RNA binding and ATP hydrolysis define key steps for Nup159 and Gle1.
abstract: Nuclear export of messenger RNA (mRNA) occurs by translocation of mRNA/protein complexes (mRNPs) through nuclear pore complexes (NPCs). The DEAD-box protein Dbp5 mediates export by triggering removal of mRNP proteins in a spatially controlled manner. This requires Dbp5 interaction with Nup159 in NPC cytoplasmic filaments and activation of Dbp5's ATPase activity by Gle1 bound to inositol hexakisphosphate (IP(6)). However, the precise sequence of events within this mechanism has not been fully defined. Here we analyze dbp5 mutants that alter ATP binding, ATP hydrolysis, or RNA binding. We found that ATP binding and hydrolysis are required for efficient Dbp5 association with NPCs. Interestingly, mutants defective for RNA binding are dominant-negative (DN) for mRNA export in yeast and human cells. We show that the DN phenotype stems from competition with wild-type Dbp5 for Gle1 at NPCs. The Dbp5-Gle1 interaction is limiting for export and, importantly, can be independent of Nup159. Fluorescence recovery after photobleaching experiments in yeast show a very dynamic association between Dbp5 and NPCs, averaging <1 sec, similar to reported NPC translocation rates for mRNPs. This work reveals critical steps in the Gle1-IP(6)/Dbp5/Nup159 cycle, and suggests that the number of remodeling events mediated by a single Dbp5 is limited.
authors.affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
authors.forename: Christine A
authors.initials: CA
authors.lastname: Hodge
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Tran
authors.forename: Kristen N
authors.initials: KN
authors.lastname: Noble
authors.forename: Abel R
authors.initials: AR
authors.lastname: Alcazar-Roman
authors.forename: Rakefet
authors.initials: R
authors.lastname: Ben-Yishay
authors.forename: John J
authors.initials: JJ
authors.lastname: Scarcelli
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Folkmann
authors.forename: Yaron
authors.initials: Y
authors.lastname: Shav-Tal
authors.forename: Susan R
authors.initials: SR
authors.lastname: Wente
authors.forename: Charles N
authors.initials: CN
authors.lastname: Cole
mesh_terms: D021581:Active Transport, Cell Nucleus; D000255:Adenosine Triphosphate / Q000378:metabolism; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D053487:DEAD-box RNA Helicases / Q000235:genetics* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D009154:Mutation; D022022:Nuclear Pore / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism; D029543:Nucleocytoplasmic Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D010641:Phenotype; D011485:Protein Binding / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: C101408:GLE1 protein, S cerevisiae; C093510:NUP159 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D029543:Nucleocytoplasmic Transport Proteins; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; C492382:DBP5 protein, S cerevisiae; C121148:DDX19B protein, human; D053487:DEAD-box RNA Helicases