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Identification, molecular cloning, and characterization of subunit 11 of the human 26S proteasome
L Hoffman1, C Gorbea, M Rechsteiner
1University of Utah School of Medicine, Department of Biochemistry, Salt Lake City 84132, USA.
Insights
Researchers identified and cloned the cDNA for human subunit 11 (S11) of the 26S proteasome. This protein is the human homolog of yeast Rpn9, suggesting conserved proteasome functions.
Area of Science:
- Molecular Biology
- Proteomics
- Cell Biology
Background:
- The 26S proteasome is a crucial cellular machine for protein degradation.
- Subunit 11 (S11) is a component of the human 26S proteasome, but its function and sequence were previously uncharacterized.
Purpose of the Study:
- To sequence and clone the cDNA for human S11.
- To characterize the protein product of the S11 cDNA.
- To identify potential functional motifs and homologs of human S11.
Main Methods:
- Peptide sequencing of purified human S11.
- cDNA cloning and sequencing.
- In vitro translation and SDS-PAGE analysis.
- Antibody production and Western blot analysis.
Main Results:
- The human S11 cDNA encodes a 376 amino acid protein (42.9 kDa, pI 5.6).
- In vitro translated S11 co-migrated with native S11 on SDS-PAGE.
- Antiserum against recombinant S11 recognized the protein in human and rabbit proteasomes.
- No functional motifs were identified in the S11 sequence.
Conclusions:
- Human S11 is a validated subunit of the 26S proteasome.
- Human S11 is the homolog of yeast Rpn9, indicating conserved proteasome structure and potentially function.
- The lack of identifiable functional motifs suggests its role may be structural or regulatory within the proteasome complex.
Abstract:
We sequenced five peptides from subunit 11 (S11), a 43 kDa protein of the human 26S proteasome, and used this information to clone its cDNA. The S11 cDNA encodes a 376 amino acid protein with a pI of 5.6 and a molecular mass of 42.9 kDa. Translation of S11 RNA in the presence of [35S]methionine produces a radiolabeled protein that co-migrates with S11 of the human 26S proteasome on SDS-PAGE. Polyclonal antiserum made against recombinant S11 recognizes a protein of the same size in extracts of bacteria expressing S11 and in purified 26S proteasomes from human red blood cells or rabbit reticulocytes. The S11 sequence does not contain motifs that suggest a biological function. S11 is, however, the human homolog of Rpn9, a recently identified subunit of the yeast 26S proteasome.