Evidence for the existence of a novel mechanism for the nuclear import of Hsc70.
Hsc70 is a multifunctional protein that is capable of shuttling between the nucleus and the cytoplasm. In this study we investigated the signal-mediated nuclear import step, using Xenopus oocytes as a model system. Purified rat hsc70, and hybrid proteins, which contained either the full-length or the mutated forms of rat hsc70 fused with the maltose binding protein, were labeled with 125I and used as transport substrates. In competition experiments, it was found that the nuclear import of neither purified hsc70 nor the full-length fusion protein was inhibited by an excess of SV40 large T or nucleoplasmin nuclear localization signals (NLSs) that were conjugated with BSA. Since hsc70 contains only a single basic domain (246KRKHKKDISENKRAVRR262), which has the characteristics of an NLS, we examined its role in nuclear import. It was determined, by conjugating this sequence with BSA, that it is capable of promoting nuclear import and, therefore, acts as a prototypical basic NLS. However, inactivation of this signal by deleting the first six amino acids (246KRKHKK251) had no effect on hsc70 import. Overall, the present results indicate that hsc70 utilizes a novel import signal and enters the nucleus by a different mechanism than that employed by simple and bipartite NLSs. The novel signal has not been identified, but we have obtained evidence that it is located in the amino terminus of hsc70.
Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville 32610, USA.
V
V
Lamian
G M
GM
Small
C M
CM
Feldherr
D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D005260:Female; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014982:Xenopus laevis
D000952:Antigens, Polyomavirus Transforming; D002352:Carrier Proteins; D017931:DNA Primers; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494373:Hspa8 protein, rat; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins