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Volumn 284, Issue 5414, 1999, Pages 662-665

Reconstitution of G1 cyclin ubiquitination with complexes containing SCF(Grr1) and Rbx1

Author keywords

[No Author keywords available]

Indexed keywords

CELL PROTEIN; CYCLINE; PROTEIN RBX1; PROTEIN SCF; REGULATOR PROTEIN; UNCLASSIFIED DRUG; ANAPHASE PROMOTING COMPLEX; ANAPHASE-PROMOTING COMPLEX; CARRIER PROTEIN; CDC53 PROTEIN, S CEREVISIAE; CELL CYCLE PROTEIN; CLN1 PROTEIN, YEAST; CULLIN; FUNGAL PROTEIN; GRR1 PROTEIN, S CEREVISIAE; HYBRID PROTEIN; LIGASE; PEPTIDE SYNTHASE; S PHASE KINASE ASSOCIATED PROTEIN; SACCHAROMYCES CEREVISIAE PROTEIN; UBIQUITIN; UBIQUITIN PROTEIN LIGASE;

EID: 0033596977     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.284.5414.662     Document Type: Article
Times cited : (359)

References (36)
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    • Yeast extracts from isogenic YM4575 (GRR1) or YM4576 (grr1Δ) strains (32) or CB018 (pep4::HIS3) were fractionated on DEAE-Sephacryl (33). Proteins editing with 250 and 500 mM KCl were collected (F250 and F500, respectively). Immunoblot analysis revealed Cdc34 only in the F500 fraction. Twenty-microliter ubiquitination reactions (30 to 60 min, 25°C) contained 500 nM Cdc34, 300 nM E1, 2 mM adenosine triphosphate (ATP), an ATP-regenerating system, 20 μM ubiquitin, 0 to 120 μg of F250, and 50 ng of active or kinase-defective (K-) HA-Cln1/GST-Cdc28 (12). Cln1 autophosphorylation was performed at 25°C in the presence of 1 mM ATP for 30 min. Reaction mixtures were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and products were visualized by immunoblotting with ECL detection (Amersham). SCF complexes were purified from insect cells 40 hours after infection with antibodies to tagged proteins (12). A baculovirus encoding untagged Grr1 (in pVL1392) was produced with Baculogold (Pharmingen). Rabbit anti-Grr1 was generated with the peptide MDQDNNNHND-SNRL (see legend to Fig. 4 for amino acid abbreviations). RBX1 and rbx1-1 (16) extracts were prepared from cells shifted to 38°C for 16 hours. Strains (MATa RBX1::HIS3 ura3 leu2 trp1 lys2 his3Δ200 can1-100 cyh2) were maintained by a plasmid containing either a wild-type (pDK101) or mutant (pDK102) version of RBX1 in pRS314.
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    • note
    • RBX1 was disrupted by inserting the LEU2 gene into the Nsi I site in the RBX1 open reading frame in pDK100 (containing a genomic RBX1 fragment in pRS316). A Sac I-Sph I fragment from pDK100 was used to transform Y80 (MATa can1-100 ade2-1 his3-11, 15 leu 2-3, 112 trp1-1 ura3-1) carrying pDK100 to generate the disruption strain. Strains that could not grow without pDK100 were chosen, and integration of the LEU2 gene was confirmed by Southern (DNA) blot analysis. The rbx1-1 allele was generated by polymerase chain reaction mutagenesis (34) with primers flanking the open reading frame. The plasmid was rescued from a temperature-sensitive strain, retested, and sequenced.
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    • note
    • Baculoviruses encoding Rbx1 and MYC3-Rbx1 [in pVL1392 or pHI-100 by universal plasmid subcloning (35), respectively] were generated with Baculogold (Pharmingen).
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    • note
    • RA), and 50 ng of HA-Cln1/GST-Cdc28. Reaction mixtures were separated by SDS-PAGE and immunoblotted.
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    • note
    • F250 (1 mg) was fractionated over a Superdex 200 column (Pharmacia) in 50 mM tris-HCl (pH 7.5), 50 mM KCL Pools of four fractions (0.25 ml each) were concentrated with Centricon-10 and samples (50 μg of protein) were used for assays (13).
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    • note
    • We thank M. Goebl for anti-Cdc34 and discussions, M. Tyers for Cdc53, M. Johnson for strains, C. Correll and R. Deshaies for HA-Grr1, and M. Li and P. Sen for technical assistance. Supported by NIH grants AG11085 (to J.W.H. and S.J.E.), GM54137 (to J.W.H.), GM41628 (to R.C.C.), the Welch Foundation (J.W.H), the H. A. and Mary K. Chapman Charitable Trust (J.W.C), and the Helen Hay Whitney Foundation (D.M.K). S.J.E. and J.W.C are Investigators with the Howard Hughes Medical Institute.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.