ABSTRACT
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.
Subject(s)
Cell Nucleus/genetics , Genomic Instability , Mitochondria/metabolism , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Cellular Senescence , Humans , Iron/metabolism , Iron-Sulfur Proteins/metabolism , Loss of Heterozygosity , Membrane Potential, Mitochondrial , Transcription, GeneticABSTRACT
Septins are GTP-binding proteins that assemble into hetero-oligomers. They can interact with each other end-to-end to form filaments, making them the fourth element of the cytoskeleton. To update the current knowledge on the ever-increasing implications of these fascinating proteins in cellular functions, a hundred expert scientists from across the globe gathered from 12 to 15 October 2021 in Berlin for the first hybrid-format (on site and virtual) EMBO workshop Molecular and Cell Biology of Septins.
Subject(s)
GTP-Binding Proteins , Septins , Berlin , Cytoskeleton/metabolism , GTP-Binding Proteins/metabolism , Microtubules/metabolism , Septins/genetics , Septins/metabolismABSTRACT
Alzheimer's disease (AD) is responsible for 60%-80% of identified cases of dementia. While the generation and accumulation of amyloid precursor protein (APP) fragments is accepted as a key step in AD pathogenesis, the precise role of these fragments remains poorly understood. To overcome this deficit, we induced the expression of the soluble C-terminal fragment of APP (C99), the rate-limiting peptide for the generation of amyloid fragments, in yeast that contain thermosensitive mutations in genes encoding proteasome subunits. Our previous work with this system demonstrated that these proteasome-deficient yeast cells, expressing C99 when proteasome activity was blunted, generated amyloid fragments similar to those observed in AD patients. We now report the phenotypic repercussions of inducing C99 expression in proteasome-deficient cells. We show increased levels of protein aggregates, cellular stress and chaperone expression, electron-dense accumulations in the nuclear envelope/ER, abnormal DNA condensation, and an induction of apoptosis. Taken together, these findings suggest that the generation of C99 and its associated fragments in yeast cells with compromised proteasomal activity results in phenotypes that may be relevant to the neuropathological processes observed in AD patients. These data also suggest that this yeast model should be useful for testing therapeutics that target AD-associated amyloid, since it allows for the assessment of the reversal of the perturbed cellular physiology observed when degradation pathways are dysfunctional.
Subject(s)
Alzheimer Disease , Proteasome Endopeptidase Complex , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Humans , Peptide Fragments/genetics , Peptide Fragments/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolismABSTRACT
How nonspore haploid Saccharomyces cells choose sites of budding and polarize towards pheromone signals in order to mate has been a subject of intense study. Unlike nonspore haploids, sibling spores produced via meiosis and sporulation by a diploid cell are physically interconnected and encased in a sac derived from the old cell wall of the diploid, called the ascus. Nonspore haploids bud adjacent to previous sites of budding, relying on stable cortical landmarks laid down during prior divisions, but because spore membranes are made de novo, it was assumed that, as is known for fission yeast, Saccharomyces spores break symmetry and polarize at random locations. Here, we show that this assumption is incorrect: Saccharomyces cerevisiae spores are born prepolarized to outgrow, prior to budding or mating, away from interspore bridges. Consequently, when spores bud within an intact ascus, their buds locally penetrate the ascus wall, and when they mate, the resulting zygotes adopt a unique morphology reflective of repolarization towards pheromone. Long-lived cortical foci containing the septin Cdc10 mark polarity sites, but the canonical bud site selection programme is dispensable for spore polarity, thus the origin and molecular composition of these landmarks remain unknown. These findings demand further investigation of previously overlooked mechanisms of polarity establishment and local cell wall digestion and highlight how a key step in the Saccharomyces life cycle has been historically neglected.
Subject(s)
Cell Wall/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/physiology , Spores, Fungal/growth & development , Spores, Fungal/genetics , GTP Phosphohydrolases/genetics , Meiosis/genetics , Membrane Proteins/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Septins/genetics , Septins/metabolism , Spores, Fungal/physiologyABSTRACT
Autophagy is a conserved cellular degradation pathway wherein double-membrane vesicles called autophagosomes capture long-lived proteins, and damaged or superfluous organelles, and deliver them to the lysosome for degradation. Septins are conserved GTP-binding proteins involved in many cellular processes, including phagocytosis and the autophagy of intracellular bacteria, but no role in general autophagy was known. In budding yeast, septins polymerize into ring-shaped arrays of filaments required for cytokinesis. In an unbiased genetic screen and in subsequent targeted analysis, we found autophagy defects in septin mutants. Upon autophagy induction, pre-assembled septin complexes relocalized to the pre-autophagosomal structure (PAS) where they formed non-canonical septin rings at PAS. Septins also colocalized with autophagosomes, where they physically interacted with the autophagy proteins Atg8 and Atg9. When autophagosome degradation was blocked in septin-mutant cells, fewer autophagic structures accumulated, and an autophagy mutant defective in early stages of autophagosome biogenesis (atg1Δ), displayed decreased septin localization to the PAS. Our findings support a role for septins in the early stages of budding yeast autophagy, during autophagosome formation.This article has an associated First Person interview with the first author of the paper.
Subject(s)
Autophagy-Related Protein 8 Family/genetics , Autophagy-Related Proteins/genetics , Autophagy/genetics , Membrane Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Septins/genetics , Autophagosomes/metabolism , Autophagy-Related Protein 8 Family/chemistry , Autophagy-Related Proteins/chemistry , Cytokinesis/genetics , Cytoskeleton/genetics , Cytoskeleton/metabolism , GTP-Binding Proteins/chemistry , GTP-Binding Proteins/genetics , Membrane Proteins/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Septins/chemistryABSTRACT
The septins are a family of GTP-binding proteins that form cytoskeletal filaments. Septins are highly conserved and evolutionarily ancient but are absent from land plants. The synthetic plant cytokinin forchlorfenuron (FCF) was shown previously to inhibit budding yeast cell division and induce ectopic septin structures (M. Iwase, S. Okada, T. Oguchi, and A. Toh-e, Genes Genet. Syst. 79:199-206, 2004, http://dx.doi.org/10.1266/ggs.79.199). Subsequent studies in a wide range of eukaryotes have concluded that FCF exclusively inhibits septin function, yet the mechanism of FCF action in nonplant cells remains poorly understood. Here, we report that the cellular effects of FCF are far more complex than previously described. The reported growth arrest of budding yeast cells treated with 1 mM FCF partly reflects sensitization caused by a bud4 mutation present in the W303 strain background. In wild-type (BUD4(+)) budding yeast, growth was inhibited at FCF concentrations that had no detectable effect on septin structure or function. Moreover, FCF severely inhibited the proliferation of fission yeast cells, in which septin function is nonessential. FCF induced fragmentation of budding yeast mitochondrial reticula and the loss of mitochondrial membrane potential. Mitochondria also fragmented in cultured mammalian cells treated with concentrations of FCF that previously were assumed to target septins only. Finally, FCF potently inhibited ciliation and motility and induced mitochondrial disorganization in Tetrahymena thermophila without apparent alterations in septin structure. None of these effects was consistent with the inhibition of septin function. Our findings point to nonseptin targets as major concerns when using FCF.
Subject(s)
Membrane Potential, Mitochondrial/drug effects , Phenylurea Compounds/pharmacology , Pyridines/pharmacology , Saccharomyces cerevisiae/growth & development , Schizosaccharomyces/growth & development , Septins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Line , Cell Movement/drug effects , Cell Proliferation/drug effects , Cytoskeleton/drug effects , Epithelial Cells/drug effects , GTP-Binding Proteins/genetics , Humans , Hyphae/growth & development , Mitochondria/metabolism , Mitochondria/pathology , Saccharomyces cerevisiae Proteins/genetics , Tetrahymena thermophila/drug effects , Tetrahymena thermophila/growth & developmentABSTRACT
Budding yeast septins assemble into hetero-octamers and filaments required for cytokinesis. Solvent-exposed cysteine (Cys) residues provide sites for attaching substituents useful in assessing assembly kinetics and protein interactions. To introduce Cys at defined locations, site-directed mutagenesis was used, first, to replace the native Cys residues in Cdc3 (C124 C253 C279), Cdc10 (C266), Cdc11 (C43 C137 C138), Cdc12 (C40 C278), and Shs1 (C29 C148) with Ala, Ser, Val, or Phe. When plasmid-expressed, each Cys-less septin mutant rescued the cytokinesis defects caused by absence of the corresponding chromosomal gene. When integrated and expressed from its endogenous promoter, the same mutants were fully functional, except Cys-less Cdc12 mutants (which were viable, but exhibited slow growth and aberrant morphology) and Cdc3(C124V C253V C279V) (which was inviable). No adverse phenotypes were observed when certain pairs of Cys-less septins were co-expressed as the sole source of these proteins. Cells grew less well when three Cys-less septins were co-expressed, suggesting some reduction in fitness. Nonetheless, cells chromosomally expressing Cys-less Cdc10, Cdc11, and Cdc12, and expressing Cys-less Cdc3 from a plasmid, grew well at 30°C. Moreover, recombinant Cys-less septins--or where one of the Cys-less septins contained a single Cys introduced at a new site--displayed assembly properties in vitro indistinguishable from wild-type.
Subject(s)
Cell Cycle Proteins/chemistry , Cysteine/chemistry , Saccharomyces cerevisiae/metabolism , Septins/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Mutagenesis, Site-Directed , Septins/genetics , Septins/metabolismABSTRACT
During response of budding yeast to peptide mating pheromone, the cell becomes markedly polarized and MAPK scaffold protein Ste5 localizes to the resulting projection (shmoo tip). We demonstrated before that this recruitment is essential for sustained MAPK signaling and requires interaction of a pleckstrin homology (PH) domain in Ste5 with phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] in the plasma membrane. Using fluorescently tagged high-affinity probes specific for PtdIns(4,5)P(2), we have now found that this phosphoinositide is highly concentrated at the shmoo tip in cells responding to pheromone. Maintenance of this strikingly anisotropic distribution of PtdIns(4,5)P(2), stable tethering of Ste5 at the shmoo tip, downstream MAPK activation, and expression of a mating pathway-specific reporter gene all require continuous function of the plasma membrane-associated PtdIns 4-kinase Stt4 and the plasma membrane-associated PtdIns4P 5-kinase Mss4 (but not the Golgi-associated PtdIns 4-kinase Pik1). Our observations demonstrate that PtdIns(4,5)P(2) is the primary determinant for restricting localization of Ste5 within the plasma membrane and provide direct evidence that an extracellular stimulus-evoked self-reinforcing mechanism generates a spatially enriched pool of PtdIns(4,5)P(2) necessary for the membrane anchoring and function of a signaling complex.
Subject(s)
MAP Kinase Signaling System , Phosphatidylinositol 4,5-Diphosphate/metabolism , Saccharomyces cerevisiae/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Surface Extensions/metabolism , Fluorescence Polarization , Fluorescent Dyes , Pheromones/pharmacology , Protein Precursors/pharmacology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/pharmacologyABSTRACT
Septins are conserved proteins found in hetero-oligomeric complexes that are incorporated into distinct structures during cell division and differentiation; yeast septins Cdc3, Cdc10, Cdc11, and Cdc12 form hetero-octamers and polymerize into filaments, which form a "collar" at the mother-bud neck [1]. Posttranslational modifications, nucleotide binding, and protein-protein and protein-lipid interactions influence assembly and disassembly of septin structures [2], but whether individual septins are used repeatedly to build higher-order assemblies was not known. We used fluorescence-based pulse-chase methods to visualize the fate of pre-existing (old) and newly synthesized (new) molecules of two septins, Cdc10 and Cdc12. They were recycled through multiple mitotic divisions, and old and new molecules were incorporated indistinguishably into the collar. Likewise, old and new subunits intermixed within hetero-octamers, indicating that exchange occurs at this organizational level. Remarkably, in meiosis, Cdc10 made during vegetative growth was reutilized to build sporulation-specific structures and reused again during spore germination for budding and during subsequent mitotic divisions. Although Cdc12 also persisted during sporulation, it was excluded from septin structures and replaced by another subunit, Spr3; only new Cdc12 populated the collar of germinating spores. Thus, mechanisms governing septin incorporation are specific to each subunit and to the developmental state of the cell.
Subject(s)
Cell Cycle Proteins/metabolism , Cell Division , Cytoskeletal Proteins/metabolism , GTP Phosphohydrolases/metabolism , Membrane Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Cell Cycle Proteins/genetics , Cytoskeletal Proteins/genetics , GTP Phosphohydrolases/genetics , Humans , Membrane Proteins/genetics , Multiprotein Complexes/metabolism , O(6)-Methylguanine-DNA Methyltransferase/genetics , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Spores, Fungal/growth & developmentABSTRACT
Membrane trafficking via targeted exocytosis to the Saccharomyces cerevisiae bud neck provides new membrane and membrane-associated factors that are critical for cytokinesis. It remains unknown whether yeast plasma membrane abscission, the final step of cytokinesis, occurs spontaneously following extensive vesicle fusion, as in plant cells, or requires dedicated membrane fission machinery, as in cultured mammalian cells. Components of the endosomal sorting complexes required for transport (ESCRT) pathway, or close relatives thereof, appear to participate in cytokinetic abscission in various cell types, but roles in cell division had not been documented in budding yeast, where ESCRTs were first characterized. By contrast, the septin family of filament-forming cytoskeletal proteins were first identified by their requirement for yeast cell division. We show here that mutations in ESCRT-encoding genes exacerbate the cytokinesis defects of cla4Δ or elm1Δ mutants, in which septin assembly is perturbed at an early stage in cell division, and alleviate phenotypes of cells carrying temperature-sensitive alleles of a septin-encoding gene, CDC10. Elevated chitin synthase II (Chs2) levels coupled with aberrant morphogenesis and chitin deposition in elm1Δ cells carrying ESCRT mutations suggest that ESCRTs normally enhance the efficiency of cell division by promoting timely endocytic turnover of key cytokinetic enzymes.
Subject(s)
Cytokinesis/physiology , Endosomal Sorting Complexes Required for Transport/metabolism , Saccharomycetales/metabolism , Septins/metabolism , Cytokinesis/genetics , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Endocytosis/genetics , Endocytosis/physiology , Endosomal Sorting Complexes Required for Transport/genetics , Exocytosis/genetics , Exocytosis/physiology , Mutation , Saccharomycetales/genetics , Septins/geneticsABSTRACT
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.
Subject(s)
Cell Cycle Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Microscopy, Electron , Polymers/chemistry , Polymers/metabolism , Protein Conformation , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
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.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomycetales , HSP90 Heat-Shock Proteins/metabolism , Humans , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Mutation , Protein Folding , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomycetales/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Intracellular proteins function in a complex milieu wherein small molecules influence protein folding and act as essential cofactors for enzymatic reactions. Thus protein function depends not only on amino acid sequence but also on the concentrations of such molecules, which are subject to wide variation between organisms, metabolic states, and environmental conditions. We previously found evidence that exogenous guanidine reverses the phenotypes of specific budding yeast septin mutants by binding to a WT septin at the former site of an Arg side chain that was lost during fungal evolution. Here, we used a combination of targeted and unbiased approaches to look for other cases of "chemical rescue" by naturally occurring small molecules. We report in vivo rescue of hundreds of Saccharomyces cerevisiae mutants representing a variety of genes, including likely examples of Arg or Lys side chain replacement by the guanidinium ion. Failed rescue of targeted mutants highlight features required for rescue, as well as key differences between the in vitro and in vivo environments. Some non-Arg mutants rescued by guanidine likely result from "off-target" effects on specific cellular processes in WT cells. Molecules isosteric to guanidine and known to influence protein folding had a range of effects, from essentially none for urea, to rescue of a few mutants by DMSO. Strikingly, the osmolyte trimethylamine-N-oxide rescued â¼20% of the mutants we tested, likely reflecting combinations of direct and indirect effects on mutant protein function. Our findings illustrate the potential of natural small molecules as therapeutic interventions and drivers of evolution.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Mutant Proteins , Mutation , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Septins/metabolismABSTRACT
Septins are a unique family of GTPases, which were discovered 50 years ago as essential genes for the asymmetric cell shape and division of budding yeast. Septins assemble into filamentous nonpolar polymers, which associate with distinct membrane macrodomains and subpopulations of actin filaments and microtubules. While structurally a cytoskeleton-like element, septins function predominantly as spatial regulators of protein localization and interactions. Septin scaffolds and barriers have provided a long-standing paradigm for the generation and maintenance of asymmetry in cell membranes. Septins also promote asymmetry by regulating the spatial organization of the actin and microtubule cytoskeleton, and biasing the directionality of membrane traffic. In this 50th anniversary perspective, we highlight how septins have conserved and adapted their roles as effectors of membrane and cytoplasmic asymmetry across fungi and animals. We conclude by outlining principles of septin function as a module of symmetry breaking, which alongside the monomeric small GTPases provides a core mechanism for the biogenesis of molecular asymmetry and cell polarity.
Subject(s)
Septins/metabolism , Actin Cytoskeleton , Animals , Cell Membrane/enzymology , Cell Membrane/metabolism , Cell Shape , Cytoskeleton , Eukaryota/enzymology , Humans , Microtubules , Septins/physiologyABSTRACT
Septin proteins evolved from ancestral GTPases and co-assemble into hetero-oligomers and cytoskeletal filaments. In Saccharomyces cerevisiae, five septins comprise two species of hetero-octamers, Cdc11/Shs1-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11/Shs1. Slow GTPase activity by Cdc12 directs the choice of incorporation of Cdc11 vs Shs1, but many septins, including Cdc3, lack GTPase activity. We serendipitously discovered that guanidine hydrochloride rescues septin function in cdc10 mutants by promoting assembly of non-native Cdc11/Shs1-Cdc12-Cdc3-Cdc3-Cdc12-Cdc11/Shs1 hexamers. We provide evidence that in S. cerevisiae Cdc3 guanidinium occupies the site of a 'missing' Arg side chain found in other fungal species where (i) the Cdc3 subunit is an active GTPase and (ii) Cdc10-less hexamers natively co-exist with octamers. We propose that guanidinium reactivates a latent septin assembly pathway that was suppressed during fungal evolution in order to restrict assembly to octamers. Since homodimerization by a GTPase-active human septin also creates hexamers that exclude Cdc10-like central subunits, our new mechanistic insights likely apply throughout phylogeny.
For a cell to work and perform its role, it relies on molecules called proteins that are made up of chains of amino acids. Individual proteins can join together like pieces in a puzzle to form larger, more complex structures. How the protein subunits fit together depends on their individual shapes and sizes. Many cells contain proteins called septins, which can assemble into larger protein complexes that are involved in range of cellular processes. The number of subunits within these complexes differs between organisms and sometimes even between cell types in the same organism. For example, yeast typically have eight subunits within a septin protein complex and struggle to survive when the number of septin subunits is reduced to six. Whereas other organisms, including humans, can make septin protein complexes containing six or eight subunits. However, it is poorly understood how septin proteins are able to organize themselves into these different sized complexes. Now, Johnson et al. show that a chemical called guanidinium helps yeast make complexes containing six septin subunits. Guanidinium has many similarities to the amino acid arginine. Comparing septins from different species revealed that one of the septin proteins in yeast lacks a key arginine component. This led Johnson et al. to propose that when guanidinium binds to septin at the site where arginine should be, this steers the septin protein towards the shape required to make a six-subunit complex. These findings reveal a new detail of how some species evolved complexes consisting of different numbers of subunits. This work demonstrates a key difference between complexes made up of six septin proteins and complexes which are made up of eight, which may be relevant in how different human cells adapt their septin complexes for different purposes. It may also become possible to use guanidinium to treat genetic diseases that result from the loss of arginine in certain proteins.
Subject(s)
Biopolymers/metabolism , Guanidine/pharmacology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/drug effects , Septins/metabolism , Arginine/metabolism , Hot Temperature , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistryABSTRACT
Septin proteins form hetero-oligomers that associate with membranes of specific curvatures, but the mechanism is unknown. In this issue, Cannon et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201807211) identify a single amphipathic helix that is necessary and sufficient for membrane curvature sensing by septins.
Subject(s)
Proteins , SeptinsABSTRACT
Septin family proteins are quite similar to each other both within and between eukaryotic species. Typically, multiple discrete septins co-assemble into linear heterooligomers (usually hexameric or octameric rods) with a variety of cellular functions. We know little about how incorporation of different septins confers different properties to such complexes. This issue is especially acute in human cells where 13 separate septin gene products (often produced in multiple forms arising from alternative start codons and differential splicing) are expressed in a tissue-specific manner. Based on sequence alignments and phylogenetic criteria, human septins fall into four distinct groups predictive of their interactions, that is, members of the same group appear to occupy the same position within oligomeric septin protomers, which are "palindromic" (have twofold rotational symmetry about a central homodimeric pair). Many such protomers are capable of end-to-end polymerization, generating filaments. Over a decade ago, a study using X-ray crystallography and single-particle electron microscopy deduced the arrangement within recombinant heterohexamers comprising representatives of three human septin groups-SEPT2, SEPT6, and SEPT7. This model greatly influenced subsequent studies of human and other septin complexes, including how incorporating a septin from a fourth group forms heterooctamers, as first observed in budding yeast. Two recent studies, including one in this issue of Cytoskeleton, provide clear evidence that, in fact, the organization of subunits within human septin heterohexamers and heterooctamers is inverted relative to the original model. These findings are discussed here in a broader context, including possible causes for the initial confusion.
Subject(s)
Cytoskeleton , Septins , Crystallography, X-Ray , Humans , PhylogenyABSTRACT
There is a striking link between increasing age and the incidence of cancer in humans. One of the hallmarks of cancer, genomic instability, has been observed in all types of organisms. In the yeast Saccharomyces cerevisiae, it was recently discovered that during the replicative lifespan, aging cells switch to a state of high genomic instability that persists until they die. In considering these and other recent results, we suggest that accumulation of oxidatively damaged protein in aging cells results in the loss of function of gene products critical for maintaining genome integrity. Determining the identity of these proteins and how they become damaged represents a new challenge for understanding the relationship between age and genetic instability.
Subject(s)
Genomic Instability , Loss of Heterozygosity , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/physiology , Gene Expression Regulation, Fungal , Genome, Fungal , Humans , Time FactorsABSTRACT
Despite remarkable advances in synthetic biology, the fact remains that it takes a living cell to make a new living cell. The information encoded in the genome is necessary to direct assembly of all cellular components, but it may not be sufficient. Some components (e.g. mitochondria) cannot be synthesized de novo, and instead require pre-existing templates, creating a fundamental continuity of life: if the template information is ever lost, the genomic code cannot suffice to ensure proper biogenesis. One type of information only incompletely encoded in the genome is the structures of macromolecular assemblies, which emerge from the conformations of the constituent molecules coupled with the ways in which these molecules interact. For many, if not most proteins, gene sequence is not the sole determinant of native conformation, particularly in the crowded cellular milieu. A partial solution to this problem lies in the functions of molecular chaperones, encoded by nearly all cellular genomes. Chaperones effectively restrict the ensemble of conformations sampled by polypeptides, promoting the acquisition of native, functional forms, but multiple proteins have evolved ways to achieve chaperone independence, perhaps by coupling folding with higher-order assembly. Here, I propose the existence of another solution: a novel mechanism of de novo folding in which the folding of specific proteins is templated by pre-folded molecules of a partner protein whose own folding also required similar templating. This hypothesis challenges prevailing paradigms by predicting that, in order to achieve a functional fold, some non-prion proteins require a seed passed down through generations.
Subject(s)
Multiprotein Complexes , Protein Folding , Protein Multimerization , Protein Subunits/chemistry , Protein Subunits/metabolism , Proteins/chemistry , Proteins/metabolism , Humans , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Protein BindingABSTRACT
The highly conserved family of septin proteins has important functions in cytokinesis in mitotically proliferating cells. A different form of cytokinesis occurs during gametogenesis in Saccharomyces cerevisiae, in which four haploid meiotic products become encased by prospore membrane (PSMs) and specialized, stress-resistant spore walls. Septins are known to localize in a series of structures near the growing PSM, but previous studies noted only mild sporulation defects upon septin mutation. We report that directed PSM extension fails in many septin-mutant cells, and, for those that do succeed, walls are abnormal, leading to increased susceptibility to heating, freezing, and digestion by the Drosophila gut. Septin mutants mislocalize the leading-edge protein (LEP) complex required for normal PSM and wall biogenesis, and ectopic expression of the LEP protein Ssp1 perturbs mitotic septin localization and function, suggesting a functional interaction. Strikingly, extra copies of septin CDC10 rescue sporulation and LEP localization in cells lacking Sma1, a phospholipase D-associated protein dispensable for initiation of PSM assembly and PSM curvature but required for PSM extension. These findings point to key septin functions in directing efficient membrane and cell wall synthesis during budding yeast gametogenesis.