Proteasomal Proteomics: Identification of Nucleotide-sensitive Proteasome-interacting Proteins by Mass Spectrometric Analysis of Affinity-purified Proteasomes

Verma, Rati; Chen, Stephen; Feldman, R. M. Renny; Schieltz, David; Yates, John R.; Dohmen, R. Jürgen; Deshaies, Raymond J.

doi:10.1091/mbc.11.10.3425

Cited by 518 publications

(486 citation statements)

References 45 publications

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“…In the simplest case, FtsH protease, the particle consists 6 copies of a 71 kDa subunit forming a complex of approximately 425 kDa, and in the most complex case, the proteasome, the particle consists of some 40 different subunits forming a complex of 2 MDa molecular weight [3,4]. The subunits are mostly arranged in six or seven subunit rings that stack on top of each other to…”

Section: Atp-dependent Proteasesmentioning

confidence: 99%

“…Cells contain a large number deubiquitinating enzymes (DUBs) [20] and at least two of them, Rpn11 and Ubp6, are located in the 19S regulatory particle and as such components of the proteasome [3,4,[21][22][23]. Rpn11 removes entire ubiquitin chains from the substrate by cleaving the isopeptide bond between the substrate and the first ubiquitin to recycle ubiquitin and to allow substrate's degradation [22,23].…”

Section: Substrate Targeting To Proteasesmentioning

confidence: 99%

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Protein targeting to ATP-dependent proteases

Inobe

Matouschek

2008

Current Opinion in Structural Biology

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ATP-dependent proteases control diverse cellular processes by degrading specific regulatory proteins. Understanding how these regulatory proteins are targeted to ATP-dependent proteases is of central importance to understanding their biological role as regulators. Recent work has shown that protein substrates are specifically transferred to ATP-dependent proteases through different routes. These routes can function in parallel or independently. In all of these targeting mechanisms it can be useful to separate two steps: substrate binding to the protease and initiation of degradation.To be active, newly synthesized protein chains must fold into three-dimensional structures, but regulated unfolding is also critically important in some biological processes, such as protein degradation by ATP-dependent proteases and protein translocation across membranes [1]. Unfolding is required during degradation because the proteolytic sites of the ATP-dependent proteases are sequestered deep inside the proteases' structures and accessible only through narrow openings. Similarly, unfolding is required during several translocation processes because the protein import channels in some organelles are not wide enough for native proteins to fit through them. The mechanisms of unfolding in both types of processes are similar to each other but different from that of unfolding induced by heat or chemical denaturants. Here we discuss how the requirement for protein unfolding during degradation affects the way ATPdependent proteases select their substrates. ATP-dependent proteasesATP-dependent proteases degrade short-lived regulatory proteins and thereby control cellular processes such as signal transduction, cell cycle, and gene transcription. The proteases also clear misfolded and aggregated proteins from the cell and produce some of the peptides to be displayed at cell surface as part of adaptive immune response. In eukaryotes, these functions are performed mainly by the proteasome. In prokaryotes and the organelles of eukaryotes, the functions are fulfill by analogues of the proteasome, such as the ClpAP, ClpXP, HslUV, FtsH, and Lon proteases. Although ATP-dependent proteases show only relatively little sequence identity, they share a common architecture [2].The ATP-dependent proteases all form large multisubunit particles (Figure 1). In the simplest case, FtsH protease, the particle consists 6 copies of a 71 kDa subunit forming a complex of approximately 425 kDa, and in the most complex case, the proteasome, the particle consists of some 40 different subunits forming a complex of 2 MDa molecular weight [3,4]. The subunits are mostly arranged in six or seven subunit rings that stack on top of each other to

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Section: Atp-dependent Proteasesmentioning

confidence: 99%

Section: Substrate Targeting To Proteasesmentioning

confidence: 99%

Protein targeting to ATP-dependent proteases

Inobe

Matouschek

2008

Current Opinion in Structural Biology

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“…18, May 2007May 1957 the proteasome can be ubiquitinated (Peng et al, 2003) and the proteasome has been suggested to undergo rapid cycles of assembly/disassembly (Babbitt et al, 2005), we first investigated a possible role for polyubiquitination in the maintenance of fully-assembled 26S proteasome complexes. When the chromosomal locus encoding Pre1, an alpha subunit of the 20S core, is replaced by an allele tagged with a Flag epitope, it is possible to obtain intact, active 26S proteasome complexes by a singlestep affinity purification method (Verma et al, 2000). Proteasomes affinity-purified from uba1-204 cells cultured at either 25 or 37°C were found to contain all 20S and 19S subunits with no detectible change in subunit composition ( Figure 4A).…”

mentioning

confidence: 99%

A Conditional Yeast E1 Mutant Blocks the Ubiquitin–Proteasome Pathway and Reveals a Role for Ubiquitin Conjugates in Targeting Rad23 to the Proteasome

Ghaboosi

Deshaies

2007

MBoC

Self Cite

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E1 ubiquitin activating enzyme catalyzes the initial step in all ubiquitin-dependent processes. We report the isolation of uba1-204, a temperature-sensitive allele of the essential Saccharomyces cerevisiae E1 gene, UBA1. Uba1-204 cells exhibit dramatic inhibition of the ubiquitin-proteasome system, resulting in rapid depletion of cellular ubiquitin conjugates and stabilization of multiple substrates. We have employed the tight phenotype of this mutant to investigate the role ubiquitin conjugates play in the dynamic interaction of the UbL/UBA adaptor proteins Rad23 and Dsk2 with the proteasome. Although proteasomes purified from mutant cells are intact and proteolytically active, they are depleted of ubiquitin conjugates, Rad23, and Dsk2. Binding of Rad23 to these proteasomes in vitro is enhanced by addition of either free or substrate-linked ubiquitin chains. Moreover, association of Rad23 with proteasomes in mutant and wild-type cells is improved upon stabilizing ubiquitin conjugates with proteasome inhibitor. We propose that recognition of polyubiquitin chains by Rad23 promotes its shuttling to the proteasome in vivo. INTRODUCTIONActivation of ubiquitin by ubiquitin-activating enzyme (E1) is the requisite first step in all ubiquitin-dependent pathways, including regulated proteolysis. The process begins with an ATP-dependent reaction in which the ubiquitin moiety forms a high-energy thioester bond with the active site cysteine of E1. E1 can then transfer the activated ubiquitin to a conjugating enzyme (E2), which acts alone or in conjunction with a ubiquitin ligase (E3) to covalently link ubiquitin to lysine residues on specific target proteins (Haas and Siepmann, 1997). Through successive ligation reactions, a polyubiquitin chain can form on the substrate and serve as a signal for targeting to the multisubunit 26S proteasome. Composed of a cylindrical 20S proteolytic core complex capped at one or both ends by 19S regulatory complexes, the proteasome deubiquitinates and unfolds substrates before translocating them into its core for proteolysis (Amerik and Hochstrasser, 2004;Pickart and Cohen, 2004).The existence of mammalian cell lines that carry temperature-sensitive alleles of E1 has been of great importance to the study of the functions of the ubiquitin system in animal cells Finley et al., 1984;Kulka et al., 1988;Salvat et al., 2000). Ironically, no tight, rapid-acting conditional mutation has been described for the budding yeast UBA1 gene that encodes E1 despite the availability of sophisticated molecular genetic techniques in this organism.Although a temperature-sensitive allele of UBA1 exists, this allele results in only moderate stabilization of tested substrates and possible defects in ubiquitination were not examined (McGrath, 1991;Gandre and Kahana, 2002). A second allele resulting from a transposon insertion upstream of the UBA1 coding sequence reduced wild-type Uba1 protein function, causing inefficient degradation of some proteins (Swanson and Hochstrasser, 2000). Additional alleles were later en...

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“…An obvious approach to break through this problem is a large-scale physical analysis of the proteasome-interacting proteins (Verma et al 2000). We are attempting to solve the same problem in a different way.…”

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confidence: 99%

Nob1p is required for biogenesis of the 26S proteasome and degraded upon its maturation in Saccharomyces cerevisiae

Tone¹,

Toh‐e²

2002

Genes Dev.

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Nob1p is a nuclear protein that forms a complex with the 19S regulatory particle of the 26S proteasome and with uncharacterized nuclear protein Pno1p. Overexpression of NOB1 overrode the defects in maturation of the 20S proteasome of ump1⌬ cells, and temperature-sensitive nob1 and pno1 mutants exhibited defects in the processing of the ␤ subunits and in the assembly of the 20S and the 26S proteasomes. A defect in either NOB1 or PNO1 caused accumulation of newly formed Pre6p in the cytoplasm, whereas Pre6p of the ump1⌬ strain accumulated in the nucleus irrespective of the temperature. Here we present a model proposing that (1) Nob1p serves as a chaperone to join the 20S proteasome with the 19S regulatory particle in the nucleus and facilitates the maturation of the 20S proteasome and degradation of Ump1p, and (2) Nob1p is then internalized into the 26S proteasome and degraded to complete 26S proteasome biogenesis.

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Proteasomal Proteomics: Identification of Nucleotide-sensitive Proteasome-interacting Proteins by Mass Spectrometric Analysis of Affinity-purified Proteasomes

Cited by 518 publications

References 45 publications

Protein targeting to ATP-dependent proteases

Protein targeting to ATP-dependent proteases

A Conditional Yeast E1 Mutant Blocks the Ubiquitin–Proteasome Pathway and Reveals a Role for Ubiquitin Conjugates in Targeting Rad23 to the Proteasome

Nob1p is required for biogenesis of the 26S proteasome and degraded upon its maturation in Saccharomyces cerevisiae

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