Arabidopsis MBP 1 gene encodes a conserved ubiquitin recognition component of the 26 S proteasome ( protein degradation / proteolysis inhibitor / protein interaction / human ) STEVEN

Multiubiquitin chain attachment is a key step leading to the selective degradation of abnormal polypeptides and many important regulatory proteins by the eukaryotic 26S proteasome. However, the mechanism by which the 26S complex recognizes this posttranslational modification is unknown. Using synthetic multiubiquitin chains to probe an expression library for interacting proteins, we have isolated an Arabidopsis cDNA, designated MBPI, that encodes a 41-kDa acidic protein exhibiting high affinity for chains, especially those containing four or more ubiquitins. Based on similar physical and immunological properties, multiubiquitin binding affinities, and peptide sequence, MBP1 is homologous to subunit 5a of the human 26S proteasome. Structurally related proteins also exist in yeast, Caenorhabditis, and other plant species. Given their binding properties, association with the 26S proteasome, and widespread distribution, MBP1, S5a, and related proteins likely function as essential ubiquitin recognition components of the 26S proteasome. The ubiquitin-dependent proteolytic pathway is a major route for the elimination of short-lived and dysfunctional proteins in eukaryotes (1, 2). Natural substrates of this pathway include important cellular regulators such as the plant photoreceptor phytochrome A (3), cyclins (4), p53 and c-Jun oncoproteins (5, 6), the yeast MATa2 transcriptional regulator and Ga protein Gpal (7, 8), and components of the NF-KB transcriptional complex (9). Degradation of various targets by the ubiquitin pathway is initiated by the covalent attachment of a chain of multiple ubiquitins to the target (10). It is accomplished by an ATP-dependent cascade of reactions involving the sequential action of multiple enzyme families: Els, E2s, and sometimes E3s-the latter two enzymes appear responsible for the specificity of the system. Conjugation results in the formation of a ubiquitin-protein adduct where a multiubiquitin chain, linked internally via an isopeptide bond through the C-terminal Gly-76 of one ubiquitin and a lysine of an adjacent ubiquitin, is attached via its free C-terminal Gly-76 to lysyl s-amino groups within the target. Once formed, multiubiquitinated proteins are selectively recognized and then degraded by the 26S proteasome, an '1500-kDa ATP-dependent proteolytic complex (1, 11, 12). The 26S proteasome is composed of two subcomplexes, the 20S proteasome (or multicatalytic protease), which contains the catalytic core of the protease, and the 19S regulatory complex, which is responsible for the ATP dependence and specificity toward ubiquitinated substrates. The 26S proteasome degrades the target protein into small peptides but releases ubiquitin in a free, functional form. In this way, ubiquitin serves as a reusable signal for protein breakdown. Although many of the -30 potential subunits of the 26S proteasome have been identified, it is not known which of these The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. is the essential factor(s) involved in the specific recognition of ubiquitin-protein conjugates (11, 12). Previous studies showed that a single 50-kDa polypeptide [designated S5a (subunit 5a of 26S protease)] present in highly purified preparations of human 26S proteasome could bind ubiquitin-lysozyme conjugates, even after the proteasome preparations were subjected to SDS/PAGE and immobilized on membranes (13). In light of the high degree of conservation of 26S subunits between plants and vertebrates (14), we exploited this finding to search for cDNAs encoding a plant homolog of human S5a by protein interaction screening of anArabidopsis thaliana cDNA expression library. Here, we describe a gene, designated MBPI,11 that encodes a protein exhibiting high affinity for multiubiquitin chains in vitro. By various criteria, MBP1 and S5a appear to be interspecific homologs. These proteins likely act as key multiubiquitin chain recognition components of the 26S proteasome. MATERIALS AND METHODS Cloning and Molecular Characterization of Arabidopsis MBPI. Multiubiquitin chains were synthesized from bovine ubiquitin (Sigma) as described (15, 16) and individually purified (17). Chains containing between 4 and 7 ubiquitin units were pooled and radiolabeled with carrier-free Na125I (0.5 mCi; 1 Ci = 37 GBq; Amersham) using lodo-Beads (Pierce). A. thaliana MBP1 was identified in an amplified, A ZAP II cDNA library prepared with 1-2 kb of poly(A)+ RNA isolated from 3-day-old, etiolated, hypocotyl/cotyledon tissue (ecotype Columbia) pretreated with ethylene (generous gift of Joe Ecker, University of Pennsylvania, Philadelphia). Approximately 2 x 105 recombinant phage were induced and protein was transferred to nitrocellulose membranes (18). Membranes were washed in Tris-buffered saline [TBS; 20mM Tris HCl, pH 7.5 (25°C)/0.5 M NaCl], blocked for 2 h in TBS containing 10 mg of bovine serum albumin (BSA) per ml, washed briefly in TBS, and incubated for 1 h in TBS containing 10 mg of BSA per ml and 3.5 x 105 cpm per ml of labeled multiubiquitin chains. Phage corresponding to positive plaques were rescued to phagemids in Escherichia coli strain XL-1 Blue MRF' (Stratagene). Complete nucleic acid sequence was determined from both DNA strands by the dideoxynucleotide chaintermination procedure with single-stranded DNA templates (19). Nucleotide and amino acid sequence manipulations employed programs of the University of Wisconsin Genetics Computer Group software package (20). DNA and RNA Gel Blot Analyses. Genomic DNA and total RNA were isolated fromArabidopsis, ecotype Columbia, using Qiagen (Chatsworth, CA) nucleic acid purification columns Abbreviation: S5a, subunit Sa of 26S protease. tS.v.N. and Q.D. contributed equally to this work. §To whom reprint requests should be addressed. IThe sequence reported in this paper has been GenBank data base (accession no. U33269). deposited in the