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Biomedical subjects

Daniel Finley

Publications and source records attributed to Daniel Finley.

8 recordsLinked to original sources
PubMed2003

A proteomics approach to understanding protein ubiquitination.

There is a growing need for techniques that can identify and characterize protein modifications on a large or global scale. We report here a proteomics approach to enrich, recover, and identify ubiquitin conjugates from Saccharomyces cerevisiae lysate. Ubiquitin conjugates from a strain expressing 6xHis-tagged ubiquitin were isolated, proteolyzed with trypsin and analyzed by multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for amino acid sequence determination. We identified 1,075 proteins from the sample. In addition, we detected 110 precise ubiquitination sites present in 72 ubiquitin-protein conjugates. Finally, ubiquitin itself was found to be modified at seven lysine residues providing evidence for unexpected diversity in polyubiquitin chain topology in vivo. The methodology described here provides a general tool for the large-scale analysis and characterization of protein ubiquitination.

Amino Acid Sequence↗
PubMed2003

Proteasome disassembly and downregulation is correlated with viability during stationary phase.

During prolonged starvation, yeast cells enter a stationary phase (SP) during which the synthesis of many proteins is dramatically decreased. We show that a parallel decrease in proteasome-dependent proteolysis also occurs. The reduction in proteolysis is correlated with disassembly of 26S proteasome holoenzymes into their 20S core particle (CP) and 19S regulatory particle (RP) components. Proteasomes are reassembled, and proteolysis resumes prior to cell cycle reentry. Free 20S CPs are found in an autoinhibited state in which the N-terminal tails from neighboring alpha subunits are anchored by an intricate lattice of interactions blocking the channel that leads into the 20S CPs. By deleting channel gating residues of CP alpha subunits, we generated an "open channel" proteasome that exhibits faster rates of protein degradation both in vivo and in vitro, indicating that gating contributes to regulation of proteasome activity. This open channel mutant is delayed in outgrowth from SP and cannot survive following prolonged starvation. In summary, we have found that the ubiquitin-proteasome pathway can be subjected to global downregulation, that the proteasome is a target of this regulation, and that proteasome downregulation is linked to survival of SP cells. Maintaining high viability during SP is essential for evolutionary fitness, which may explain the extreme conservation of channel gating residues in eukaryotic proteasomes.

Caseins↗
PubMed2002

Rpn6p, a proteasome subunit from Saccharomyces cerevisiae, is essential for the assembly and activity of the 26 S proteasome.

We report the functional characterization of RPN6, an essential gene from Saccharomyces cerevisiae encoding the proteasomal subunit Rpn6p. For this purpose, conditional mutants that are able to grow on galactose but not on glucose were obtained. When these mutants are shifted to glucose, Rpn6p depletion induces several specific phenotypes. First, multiubiquitinated proteins accumulate, indicating a defect in proteasome-mediated proteolysis. Second, mutant yeasts are arrested as large budded cells with a single nucleus and a 2C DNA content; in addition, the spindle pole body is duplicated, indicating a general cell cycle defect related to the turnover of G(2)-cyclins after DNA synthesis. Clb2p and Pds1p, but not Sic1p, accumulate in the arrested cells. Depletion of Rpn6p affects both the structure and the peptidase activity of proteasomes in the cell. These results implicate Rpn6p function in the specific recognition of a subset of substrates and point to a role in maintaining the correct quaternary structure of the 26 S proteasome.

CDC28 Protein Kinase, S cerevisiae↗
PubMed2002

Role of a ubiquitin-like modification in polarized morphogenesis.

Type I ubiquitin-like proteins constitute a family of protein modifiers. Here we report the identification of a posttranslational protein modifier from Saccharomyces cerevisiae, Hub1. Overexpression of Hub1 resulted in enhanced conjugate formation when its carboxyl-terminal residue was deleted, suggesting that mature Hub1 may be produced by proteolytic processing. In vivo targets of Hub1 conjugation included cell polarity factors Sph1 and Hbt1. In the hub1Delta mutant, the subcellular localization of both Hbt1 and Sph1 was disrupted, and cell polarization during the formation of mating projections was defective. Consistent with these polarization defects, the hub1Delta mutant was deficient in mating.

Amino Acid Sequence↗
PubMed2002

Multiple associated proteins regulate proteasome structure and function.

We have identified proteins that are abundant in affinity-purified proteasomes, but absent from proteasomes as previously defined because elevated salt concentrations dissociate them during purification. The major components are a deubiquitinating enzyme (Ubp6), a ubiquitin-ligase (Hul5), and an uncharacterized protein (Ecm29). Ecm29 tethers the proteasome core particle to the regulatory particle. Proteasome binding activates Ubp6 300-fold and is mediated by the ubiquitin-like domain of Ubp6, which is required for function in vivo. Ubp6 recognizes the proteasome base and its subunit Rpn1, suggesting that proteasome binding positions Ubp6 proximally to the substrate translocation channel. ubp6Delta mutants exhibit accelerated turnover of ubiquitin, indicating that deubiquitination events catalyzed by Ubp6 prevent translocation of ubiquitin into the proteolytic core particle.

Adenosine Triphosphate↗
PubMed2002

Protein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48.

Endoplasmic reticulum (ER)-associated protein degradation by the ubiquitin-proteasome system requires the dislocation of substrates from the ER into the cytosol. It has been speculated that a functional ubiquitin proteasome pathway is not only essential for proteolysis, but also for the preceding export step. Here, we show that short ubiquitin chains synthesized on proteolytic substrates are not sufficient to complete dislocation; the size of the chain seems to be a critical determinant. Moreover, our results suggest that the AAA proteins of the 26S proteasome are not directly involved in substrate export. Instead, a related AAA complex Cdc48, is required for ER-associated protein degradation upstream of the proteasome.

Adenosine Triphosphatases↗
PubMed2002

Proteasome subunit Rpn1 binds ubiquitin-like protein domains.

The yeast protein Rad23 belongs to a diverse family of proteins that contain an amino-terminal ubiquitin-like (UBL) domain. This domain mediates the binding of Rad23 to proteasomes, which in turn promotes DNA repair and modulates protein degradation, possibly by delivering ubiquitinylated cargo to proteasomes. Here we show that Rad23 binds proteasomes by directly interacting with the base subcomplex of the regulatory particle of the proteasome. A component of the base, Rpn1, specifically recognizes the UBL domain of Rad23 through its leucine-rich-repeat-like (LRR-like) domain. A second UBL protein, Dsk2, competes with Rad23 for proteasome binding, which suggests that the LRR-like domain of Rpn1 may participate in the recognition of several ligands of the proteasome. We propose that the LRR domain of Rpn1 may be positioned in the base to allow the cargo proteins carried by Rad23 to be presented to the proteasomal ATPases for unfolding. We also report that, contrary to expectation, the base subunit Rpn10 does not mediate the binding of UBL proteins to the proteasome in yeast, although it can apparently contribute to the binding of ubiquitin chains by intact proteasomes.

Binding, Competitive↗