Interaction of yeast importin alpha with the NLS of prothymosin alpha is insufficient to trigger nuclear uptake of cargos.
A proliferation-related human protein prothymosin alpha displays exclusively nuclear localization when produced in human and Saccharomyces cerevisiae cells, whereas its isolated bipartite NLS confers nuclear targeting of the GFP reporter in human but not in yeast cells. To test whether this observation is indicative of the existence of specific requirements for nuclear targeting of proteins in yeast, a set of prothymosin alpha deletion mutants was constructed. Subcellular localization of these mutants fused to GFP was determined in yeast and compared with their ability to bind yeast importin alpha (Srp1p) in vitro. The NLS of prothymosin alpha turned out to be both necessary and sufficient to provide protein recognition by importin alpha. However, the NLS-importin alpha interaction did not ensure nuclear targeting of prothymosin alpha derivatives. This defect could be complemented by adding distinct prothymosin alpha sequences to the NLS-containing import substrate, possibly by providing binding site(s) for additional components of the yeast nuclear import machinery.
Shakulov
V R
VR
Belozersky Institute of Physico-Chemical Biology and Center of Molecular Medicine, Moscow State University, Moscow, 119899, Russia.
Vorobjev
I A
IA
Rubtsov
Y P
YP
Chichkova
N V
NV
Vartapetian
A B
AB
D000957:Antigens, Viral, Tumor / Q000378:metabolism; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism*; D002846:Chromatography, Affinity; D003593:Cytoplasm / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D028884:Karyopherins; D008164:Luminescent Proteins / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals / Q000502:physiology*; D009687:Nuclear Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion / Q000235:genetics; D013947:Thymosin / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism*
D000957:Antigens, Viral, Tumor; D005656:Fungal Proteins; D006360:Heat-Shock Proteins; D028884:Karyopherins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C491115:TIR1 protein, S cerevisiae; C040782:prothymosin alpha; D049452:Green Fluorescent Proteins; D013947:Thymosin