Abstract
Protein import into the cell nucleus requires specific binding of nuclear proteins to the nuclear pore complex. Based on amino acid sequence "motifs" of known nuclear targeting signals, we identified peptides within a number of nuclear proteins with likely nuclear targeting potential and tested their function by transfecting into cells fusion genes that produce the cytoplasmic "reporter" protein, pyruvate kinase (PK), joined to the test sequence. Sequences within c-myb (PLLKKIKQ), N-myc (PPQKKIKS), p53 (PQPKKKP), and c-erb-A (SKRVAKRKL) oncoproteins that direct PK hybrids into the nucleus were identified. A peptide (GRKKRRQRRRAP) of the human immunodeficiency virus (HIV) tat protein (Tat), which contains two short basic regions, targets fusion proteins to the nucleolus. The COOH-terminal basic Tat region (QRRRAP) does not target PK hybrid proteins into the nucleus, but mutation of two basic amino acids in this region decreases but does not abolish nucleolar accumulation mediated by the entire Tat nucleolar targeting sequence. Moreover, the c-Myc nuclear targeting sequence fused to the COOH-terminal basic Tat region (PAAKRVKLDQRRRAP) effectively localizes PK hybrids to the nucleus and nucleolus. A similar sequence (FKRKHKKDISQNKRAVRR) in the human heat-shock protein HSP70 also localizes PK to the nucleus and nucleolus.
Highlights
Protein import into thceell nucleusrequires specific 328 (PAAKRLKVD) direct a covalently linked cytoplasmic binding of nuclear proteins to the nuclear pore com- “reporter”protein,pyruvate kinase, into the nucleus complex
A syntheticpeptide containing amino acids clear targeting potential and tested their function by 320-328 can rapidly direct peptide-conjugated human serum transfecting into cells fusion genes that produce the albumin into nuclei of microinjected cells, whereas microincytoplasmic “reporter” protein, pyruvate kinase (PK), jected human serum albumin conjugated to amino acids 364
374 accumulates inthe nucleus very slowly[5].By recognizing sequence “motifs” withinthese and other known nuclear targeting peptides, we identified peptides within c-erb-A, cmyb, N-myc, p53, human immunodeficiency virus (HIV)’ tat proteins, and heat-shock protein HSP70 that specify nuclear location and that may serve as their nuclear translocation signals
Summary
Cell Culture, Transfection, and Microscopy-COS7 cells were cultured in Dulbecco's modified essential medium (DMEM, GIBCO) containing 10% fetalcalf serum (FCS) asdescribed [4,5]. Coverslips containing transfected cells were prepared for immunofluorescent microscopy as described [5]. Coverslips were washed with PBS containing 10% FCS three times and incubated with rhodamine-goat anti-rabbit antibody for 1 h, washed, and mounted for microscopy. Theserecombinant fusiongenes were transfectedinto COS7 cells as described ( 5 ) , and the subcellular location of the fusion proteins was determined by indirect immunofluorescent microscopy usinganti-PKantibody [5, 6] infour separate transfection experimentsA. 11; Fig. 1E).The fusion protein containing theerb-A sequence B peptide (Table 11) is both nuclear and cytoplasmic in the majority (80%) of transfected cells (Fig. 1A). The hybrid protein containing the c-Mysebquence displaysa nuclear and cytoplasmic distribution in abouthalf of the transfectedcells (Table 11; Fig. 1C). Some transfected cells display nucleoplasmicstaining in addition tonucleolar and cytoplasmic staining (Fig. 2F)
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