Identification of long-lived proteins retained in cells undergoing repeated asymmetric divisions.

Long-lived proteins have been implicated in age-associated decline in metazoa, but they have only been identified in extracellular matrices or postmitotic cells. However, the aging process also occurs in dividing cells undergoing repeated asymmetric divisions. It was not clear whether long-lived proteins exist in asymmetrically dividing cells or whether they are involved in aging. Here we identify long-lived proteins in dividing cells during aging using the budding yeast, Saccharomyces cerevisiae. Yeast mother cells undergo a limited number of asymmetric divisions that define replicative lifespan. We used stable-isotope pulse-chase and total proteome mass-spectrometry to identify proteins that were both long-lived and retained in aging mother cells after ∼ 18 cells divisions. We identified ∼ 135 proteins that we designate as long-lived asymmetrically retained proteins (LARPS). Surprisingly, the majority of LARPs appeared to be stable fragments of their original full-length protein. However, 15% of LARPs were full-length proteins and we confirmed several candidates to be long-lived and retained in mother cells by time-lapse microscopy. Some LARPs localized to the plasma membrane and remained robustly in the mother cell upon cell division. Other full-length LARPs were assembled into large cytoplasmic structures that had a strong bias to remain in mother cells. We identified age-associated changes to LARPs that include an increase in their levels during aging because of their continued synthesis, which is not balanced by turnover. Additionally, several LARPs were posttranslationally modified during aging. We suggest that LARPs contribute to age-associated phenotypes and likely exist in other organisms.

Disorder targets misorder in nuclear quality control degradation: a disordered ubiquitin ligase directly recognizes its misfolded substrates.

Protein quality control (PQC) degradation systems protect the cell from the toxic accumulation of misfolded proteins. Because any protein can become misfolded, these systems must be able to distinguish abnormal proteins from normal ones, yet be capable of recognizing the wide variety of distinctly shaped misfolded proteins they are likely to encounter. How individual PQC degradation systems accomplish this remains an open question. Here we show that the yeast nuclear PQC ubiquitin ligase San1 directly recognizes its misfolded substrates via intrinsically disordered N- and C-terminal domains. These disordered domains are punctuated with small segments of order and high sequence conservation that serve as substrate-recognition sites San1 uses to target its different substrates. We propose that these substrate-recognition sites, interspersed among flexible, disordered regions, provide San1 an inherent plasticity which allows it to bind its many, differently shaped misfolded substrates.

Mitochondrial dysfunction leads to nuclear genome instability via an iron-sulfur cluster defect.

Mutations and deletions in the mitochondrial genome (mtDNA), as well as instability of the nuclear genome, are involved in multiple human diseases. Here, we report that in Saccharomyces cerevisiae, loss of mtDNA leads to nuclear genome instability, through a process of cell-cycle arrest and selection we define as a cellular crisis. This crisis is not mediated by the absence of respiration, but instead correlates with a reduction in the mitochondrial membrane potential. Analysis of cells undergoing this crisis identified a defect in iron-sulfur cluster (ISC) biogenesis, which requires normal mitochondrial function. We found that downregulation of nonmitochondrial ISC protein biogenesis was sufficient to cause increased genomic instability in cells with intact mitochondrial function. These results suggest mitochondrial dysfunction stimulates nuclear genome instability by inhibiting the production of ISC-containing protein(s), which are required for maintenance of nuclear genome integrity. For a video summary of this article, see the PaperFlick file available with the online Supplemental Data.

A genetic screen for increased loss of heterozygosity in Saccharomyces cerevisiae.

Loss of heterozygosity (LOH) can be a driving force in the evolution of mitotic/somatic diploid cells, and cellular changes that increase the rate of LOH have been proposed to facilitate this process. In the yeast Saccharomyces cerevisiae, spontaneous LOH occurs by a number of mechanisms including chromosome loss and reciprocal and nonreciprocal recombination. We performed a screen in diploid yeast to identify mutants with increased rates of LOH using the collection of homozygous deletion alleles of nonessential genes. Increased LOH was quantified at three loci (MET15, SAM2, and MAT) on three different chromosomes, and the LOH events were analyzed as to whether they were reciprocal or nonreciprocal in nature. Nonreciprocal LOH was further characterized as chromosome loss or truncation, a local mutational event (gene conversion or point mutation), or break-induced replication (BIR). The 61 mutants identified could be divided into several groups, including ones that had locus-specific effects. Mutations in genes involved in DNA replication and chromatin assembly led to LOH predominantly via reciprocal recombination. In contrast, nonreciprocal LOH events with increased chromosome loss largely resulted from mutations in genes implicated in kinetochore function, sister chromatid cohesion, or relatively late steps of DNA recombination. Mutants of genes normally involved in early steps of DNA damage repair and signaling produced nonreciprocal LOH without an increased proportion of chromosome loss. Altogether, this study defines a genetic landscape for the basis of increased LOH and the processes by which it occurs.

Synthetic lethal screens identify gene silencing processes in yeast and implicate the acetylated amino terminus of Sir3 in recognition of the nucleosome core.

Dot1 methylates histone H3 lysine 79 (H3K79) on the nucleosome core and is involved in Sir protein-mediated silencing. Previous studies suggested that H3K79 methylation within euchromatin prevents nonspecific binding of the Sir proteins, which in turn facilitates binding of the Sir proteins in unmethylated silent chromatin. However, the mechanism by which the Sir protein binding is influenced by this modification is unclear. We performed genome-wide synthetic genetic array (SGA) analysis and identified interactions of DOT1 with SIR1 and POL32. The synthetic growth defects found by SGA analysis were attributed to the loss of mating type identity caused by a synthetic silencing defect. By using epistasis analysis, DOT1, SIR1, and POL32 could be placed in different pathways of silencing. Dot1 shared its silencing phenotypes with the NatA N-terminal acetyltransferase complex and the conserved N-terminal bromo adjacent homology (BAH) domain of Sir3 (a substrate of NatA). We classified all of these as affecting a common silencing process, and we show that mutations in this process lead to nonspecific binding of Sir3 to chromatin. Our results suggest that the BAH domain of Sir3 binds to histone H3K79 and that acetylation of the BAH domain is required for the binding specificity of Sir3 for nucleosomes unmethylated at H3K79.

Ubp10/Dot4p regulates the persistence of ubiquitinated histone H2B: distinct roles in telomeric silencing and general chromatin.

We previously discovered that the ubiquitin protease Ubp10/Dot4p is important for telomeric silencing through its interaction with Sir4p. However, the mechanism of Ubp10p action was unknown. We now provide evidence that Ubp10p removes ubiquitin from histone H2B; cells with UBP10 deleted have increased steady-state levels of H2B ubiquitination. As a consequence, ubp10delta cells also have increased steady-state levels of histone H3 Lys4 and Lys79 methylation. Consistent with its role in silencing, Ubp10p is preferentially localized to silent chromatin where its ubiquitin protease activity maintains low levels of H3 Lys4 and Lys79 methylation to allow optimal Sir protein binding to telomeres and global telomeric silencing. The ubiquitin protease Ubp8p has also been shown to remove ubiquitin from H2B, and ubp8delta cells have increased steady-state levels of H2B ubiquitination similar to those in ubp10delta cells. Unlike ubp10delta cells, however, ubp8delta cells do not have increased steady-state levels of H3 Lys4 and Lys79 methylation, nor is telomeric silencing affected. Despite their separate functions in silencing and SAGA-mediated transcription, respectively, deletion of both UBP10 and UBP8 results in a synergistic increase in the steady-state levels of H2B ubiquitination and in the number of genes with altered expression, indicating that Ubp10p and Ubp8p likely overlap in some of their target chromatin regions. We propose that Ubp10p and Ubp8p are the only ubiquitin proteases that normally remove monoubiquitin from histone H2B and, while there are regions of the genome to which each is specifically targeted, both combine to regulate the global balance of H2B ubiquitination.

Degradation-mediated protein quality control in the nucleus.

Protein quality control degradation systems rid the cell of aberrant proteins, preventing detrimental effects on normal cellular function. Although such systems have been identified in most subcellular compartments, none have been found in the nucleus. Here, we report the discovery of such a system in Saccharomyces cerevisiae. It is defined by San1p, a ubiquitin-protein ligase that, in conjunction with the ubiquitin-conjugating enzymes Cdc34p and Ubc1p, targets four distinct mutant nuclear proteins for ubiquitination and destruction by the proteasome. San1p has exquisite specificity for aberrant proteins and does not target the wild-type versions of its mutant substrates. San1p is nuclear localized and requires nuclear localization for function. Loss of SAN1 results in a chronic stress response, underscoring its role of protein quality control in the cell. We propose that San1p-mediated degradation acts as the last line of proteolytic defense against the deleterious accumulation of aberrant proteins in the nucleus and that analogous systems exist in other eukaryotes.