Schistosoma mansoni: Heterologous complementation of a yeast null mutant by SmRbx, a protein similar to a RING box protein involved in ubiquitination.
The SCF (Skp1-Cul1-F-box) complex is one of the several E3 ligase enzymes and it catalyzes protein ubiquitination and degradation by the 26S proteasome. Rbx1 is a member of the SCF complex in humans and HRT1 is its yeast orthologue. A cDNA encoding a Schistosoma mansoni Rbx1 homolog was cloned and functionally characterized. Heterologous functional complementation in yeast showed that the worm SmRbx gene was able to complement the HRT1yeast null mutation. Gene deletion constructs for N- and C-termini truncated proteins were used to transform hrt1(-) yeast mutant strains, allowing us to observe that regions reported to be involved in the interaction with cullin1 (Cul1) were essential for SmRbx function. Yeast two-hybrid assays using SmRbx and yeast Cul1 confirmed that SmRbx, but not the mutant SmRbxDelta24N, lacking the N-terminus of the protein, was capable of interacting with Cul1. These results suggest that SmRbx protein is involved in the SCF complex formation.
Santos
Débora N
DN
Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Pampulha, Belo Horizonte, MG 31270-901, Brazil.
Aguiar
Pedro H N
PH
Lobo
Francisco P
FP
Mourão
Marina M
MM
Tambor
José H M
JH
Valadão
Analina F
AF
Vilas-Boas
Adlane
A
Nobrega
Francisco G
FG
LoVerde
Philip T
PT
Macedo
Andréa M
AM
Pena
Sérgio D J
SD
Machado
Carlos R
CR
Franco
Glória R
GR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry; D018509:DNA, Helminth / Q000737:chemistry; D020224:Expressed Sequence Tags; D005260:Female; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D044843:SKP Cullin F-Box Protein Ligases; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D012550:Schistosoma mansoni / Q000235:genetics* / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C100318:Cullin 1; D044842:Cullin Proteins; D018076:DNA, Complementary; D018509:DNA, Helminth; D015801:Helminth Proteins; C479529:RBX1 protein, human; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C485632:HRT1 protein, S cerevisiae; D044843:SKP Cullin F-Box Protein Ligases