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.
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Septins are GTP-binding proteins that form ordered, rod-like multimeric complexes and polymerize into filaments, but how such supramolecular structure is related to septin function was unclear. In ...Saccharomyces cerevisiae, four septins form an apolar hetero-octamer (Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11) that associates end-to-end to form filaments. We show that septin filament assembly displays previously unanticipated plasticity. Cells lacking Cdc10 or Cdc11 are able to divide because the now-exposed subunits (Cdc3 or Cdc12, respectively) retain an ability to homodimerize via their so-called G interface, thereby allowing for filament assembly. In such cdc10Δ and cdc11Δ cells, the remaining septins, like wild-type complexes, localize to the cortex at the bud neck and compartmentalize nonseptin factors, consistent with a diffusion barrier composed of continuous filaments in intimate contact with the plasma membrane. Conversely, Cdc10 or Cdc11 mutants that cannot self-associate, but “cap” Cdc3 or Cdc12, respectively, prevent filament formation, block cortical localization, and kill cells.
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► In vitro septin filament formation can occur with one septin missing ► Septin homodimerization required for viability in cells lacking a different septin ► Septin localization and barrier function maintained in cells lacking one septin ► Blocking septin filament assembly disrupts localization and causes cell lethality
Septins are conserved guanosine triphosphate-binding cytoskeletal proteins involved in membrane remodeling. In budding yeast, five mitotic septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1), which are ...essential for cytokinesis, transition during bud growth from a patch to a collar, which splits into two rings in cytokinesis and is disassembled before the next cell cycle. Cdc3, Cdc10, Cdc11, and Cdc12 form an apolar octameric rod with Cdc11 at each tip, which polymerizes into straight paired filaments. We show that Shs1 substitutes for Cdc11, resulting in octameric rods that do not polymerize into filaments but associate laterally, forming curved bundles that close into rings. In vivo, half of shs1Δ mutant cells exhibit incomplete collars and disrupted neck filaments. Importantly, different phosphomimetic mutations in Shs1 can either prevent ring formation or promote formation of a gauzelike meshwork. These results show that a single alternative terminal subunit is sufficient to confer a distinctive higher-order septin ultrastructure that can be further regulated by phosphorylation.
Resting on social exchange theory and the Ability-Motivation-Opportunity framework, this research examined the causal relationships between high-performance work practices, climate for engagement, ...job engagement, and job performance in the context of Vietnamese higher education. A multiphase, multisource data collection method was applied to 394 pairs of full-time academic staff and their line managers or supervisors working for 14 public and private universities in Ho Chi Minh City, Vietnam. Using SEM and MSEM to analyze the data, the research pinpointed that of the three dimensions of high-performance work practices, ability-enhancing and opportunity-enhancing practices had significant and positive influences on Vietnamese university academic staff's job engagement. Climate for engagement was a crucial mediator in the influences of ability-enhancing and motivation-enhancing practices on Vietnamese university academic staff's job engagement. As their job engagement increased, it led to an improvement in their job performance. These significant findings explain how high-performance work practices promote Vietnamese university academic staff's job performance.
During division, certain cellular contents can be distributed unequally; daughter cells with different fates have different needs. Septins are proteins that participate in the establishment and ...maintenance of asymmetry during cell morphogenesis, thereby contributing to the unequal partitioning of cellular contents during division. The septins themselves provide a paradigm for studying how elaborate multi-component structures are assembled, dynamically modified, and segregated through each cell division cycle and during development. Here we review our current understanding of the supramolecular organization of septins, the function of septins in cellular compartmentalization, and the mechanisms that control assembly, dynamics, and inheritance of higher-order septin structures, with particular emphasis on recent findings made in budding yeast (Saccharomyces cerevisiae).
Septins are a conserved family of GTP-binding proteins that assemble into symmetric linear heterooligomeric complexes, which in turn are able to polymerize into apolar filaments and higher-order ...structures. In budding yeast (
Saccharomyces cerevisiae) and other eukaryotes, proper septin organization is essential for processes that involve membrane remodeling, such as the execution of cytokinesis. In yeast, four septin subunits form a Cdc11–Cdc12–Cdc3–Cdc10–Cdc10–Cdc3–Cdc12–Cdc11 heterooctameric rod that polymerizes into filaments thought to form a collar around the bud neck in close contact with the inner surface of the plasma membrane. To explore septin–membrane interactions, we examined the effect of lipid monolayers on septin organization at the ultrastructural level using electron microscopy. Using this methodology, we have acquired new insights into the potential effect of septin–membrane interactions on filament assembly and, more specifically, on the role of phosphoinositides. Our studies demonstrate that budding yeast septins interact specifically with phosphatidylinositol-4,5-
bisphosphate (PIP2) and indicate that the N terminus of Cdc10 makes a major contribution to the interaction of septin filaments with PIP2. Furthermore, we found that the presence of PIP2 promotes filament polymerization and organization on monolayers, even under conditions that prevent filament formation in solution or for mutants that prevent filament formation in solution. In the extreme case of septin complexes lacking the normally terminal subunit Cdc11 or the normally central Cdc10 doublet, the combination of the PIP2-containing monolayer and nucleotide permitted filament formation
in vitro via atypical Cdc12–Cdc12 and Cdc3–Cdc3 interactions, respectively.
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► PIP2-containing lipid monolayers promote a novel organization of septin filaments not observed in solution. ► PIP2-containing lipid monolayers dramatically enhance the formation of filaments, even for septin complexes lacking one of the septin proteins. ► The N terminus of Cdc10 is important for interaction with PIP2. ► Our
in vitro studies strongly suggest that the PIP2 present at the bud neck is important for septin organization
in situ.
Mitotic yeast cells express five septins (Cdc3, Cdc10, Cdc11, Cdc12, and Shs1/Sep7). Only Shs1 is nonessential. The four essential septins form a complex containing two copies of each, but their ...arrangement was not known. Single-particle analysis by EM confirmed that the heterooligomer is octameric and revealed that the subunits are arrayed in a linear rod. Identity of each subunit was determined by examining complexes lacking a given septin, by antibody decoration, and by fusion to marker proteins (GFP or maltose binding protein). The rod has the order Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 and, hence, lacks polarity. At low ionic strength, rods assemble end-to-end to form filaments but not when Cdc11 is absent or its N terminus is altered. Filaments invariably pair into long parallel "railroad tracks." Lateral association seems to be mediated by heterotetrameric coiled coils between the paired C-terminal extensions of Cdc3 and Cdc12 projecting orthogonally from each filament. Shs1 may be able to replace Cdc11 at the end of the rod. Our findings provide insights into the molecular mechanisms underlying the function and regulation of cellular septin structures.
Life requires the oligomerization of individual proteins into higher-order assemblies. In order to form functional oligomers, monomers must adopt appropriate 3D structures. Molecular chaperones ...transiently bind nascent or misfolded proteins to promote proper folding. Single missense mutations frequently cause disease by perturbing folding despite chaperone engagement. A misfolded mutant capable of oligomerizing with wild-type proteins can dominantly poison oligomer function. We previously found evidence that human-disease-linked mutations in Saccharomyces cerevisiae septin proteins slow folding and attract chaperones, resulting in a kinetic delay in oligomerization that prevents the mutant from interfering with wild-type function. Here, we build upon our septin studies to develop a new approach for identifying chaperone interactions in living cells, and use it to expand our understanding of chaperone involvement, kinetic folding delays, and oligomerization in the recessive behavior of tumor-derived mutants of the tumor suppressor p53. We find evidence of increased binding of several cytosolic chaperones to a recessive, misfolding-prone mutant, p53(V272M). Similar to our septin results, chaperone overexpression inhibits the function of p53(V272M) with minimal effect on the wild type. Unlike mutant septins, p53(V272M) is not kinetically delayed under conditions in which it is functional. Instead, it interacts with wild-type p53 but this interaction is temperature sensitive. At high temperatures or upon chaperone overexpression, p53(V272M) is excluded from the nucleus and cannot function or perturb wild-type function. Hsp90 inhibition liberates mutant p53 to enter the nucleus. These findings provide new insights into the effects of missense mutations.
Septin hetero-oligomers polymerize into cytoskeletal filaments with essential functions in many eukaryotic cell types. Mutations within the oligomerization interface that encompasses the GTP-binding ...pocket of a septin (its "G interface") cause thermoinstability of yeast septin hetero-oligomer assembly, and human disease. When coexpressed with its wild-type counterpart, a G interface mutant is excluded from septin filaments, even at moderate temperatures. We show that this quality control mechanism is specific to G interface mutants, operates during de novo septin hetero-oligomer assembly, and requires specific cytosolic chaperones. Chaperone overexpression lowers the temperature permissive for proliferation of cells expressing a G interface mutant as the sole source of a given septin. Mutations that perturb the septin G interface retard release from these chaperones, imposing a kinetic delay on the availability of nascent septin molecules for higher-order assembly. Un-expectedly, the disaggregase Hsp104 contributes to this delay in a manner that does not require its "unfoldase" activity, indicating a latent "holdase" activity toward mutant septins. These findings provide new roles for chaperone-mediated kinetic partitioning of non-native proteins and may help explain the etiology of septin-linked human diseases.
There is a strong correlation between age and cancer, but the mechanism by which this phenomenon occurs is unclear. We chose Saccharomyces cerevisiae to examine one of the hallmarks of cancer-genomic ...instability-as a function of cellular age. As diploid yeast mother cells aged, an ~100-fold increase in loss of heterozygosity (LOH) occurred. Extending life-span altered neither the onset nor the frequency of age-induced LOH; the switch to hyper-LOH appears to be on its own clock. In young cells, LOH occurs by reciprocal recombination, whereas LOH in old cells was nonreciprocal, occurring predominantly in the old mother's progeny. Thus, nuclear genomes may be inherently unstable with age.
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