The beta-amyloid precursor protein functions as a cytosolic anchoring site that prevents Fe65 nuclear translocation.
In this study we addressed the question of the intracellular localization of Fe65, an adaptor protein interacting with the beta-amyloid precursor protein (APP) and with the transcription factor CP2/LSF/LBP1. By using tagged Fe65 expression vectors, we observed that a significant fraction of Fe65 is localized in the nucleus of transfected COS7 cells. Furthermore, the isolation of nuclei from untransfected PC12 cells allowed us to observe that a part of the endogenous Fe65 is present in the nuclear extract. The analysis of Fe65 mutant constructs demonstrated that the region of the protein required for its nuclear translocation includes the WW domain, and that, on the other hand, a small fragment of 100 residues, including this WW domain, contains enough structural information to target a reporter protein (green fluorescent protein (GFP)-GFP) to the nucleus. To evaluate whether the Fe65-APP interaction could affect Fe65 intracellular trafficking, COS7 cells were cotransfected with APP(695) or APP(751) and with GFP-Fe65 expression vectors. These experiments demonstrated that Fe65 is no longer translocated to the nucleus when the cells overexpress APP, whereas the nuclear targeting of GFP-Fe65 mutants, unable to interact with APP, is unaffected by the coexpression of APP, thus suggesting that the interaction with APP anchors Fe65 in the cytosol.
Minopoli
G
G
Dipartimento di Biochimica e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, I-80131 Naples, Italy.
de Candia
P
P
Bonetti
A
A
Faraonio
R
R
Zambrano
N
N
Russo
T
T
D021581:Active Transport, Cell Nucleus; D016564:Amyloid beta-Protein Precursor / Q000502:physiology*; D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D016716:PC12 Cells; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D016564:Amyloid beta-Protein Precursor; C486242:Apbb1 protein, rat; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins