Characterization of Bud3 domains sufficient for bud neck targeting in S. cerevisiae.

The cytoskeleton serves a diverse set of functions in both multi- and unicellular organisms, including movement, transport, morphology, cell division and cell signalling. The septin family of cytoskeletal proteins are found within all fungi and metazoans and can generate three-dimensional scaffolds in vivo that promote membrane curvature, serve as physical barriers and coordinate cell cycle checkpoints. In budding yeast, the septins organize into polymerized filaments that decorate the division site between mother and daughter cells during mitosis; assembly of this structure at the 'bud neck' is critical for completion of cytokinesis and execution of numerous other cellular events. One such pathway includes bud site selection and the recruitment of proteins such as Bud4 and Bud3 that are responsible for promoting an axial budding pattern in haploid yeast. While Bud4 appears to be recruited to the septins independently of the presence of Bud3, it is likely that Bud3 can localize to the bud neck using both Bud4-dependent and Bud4-independent mechanisms. Furthermore, it remains unclear which precise domain or domains within Bud3 is/are both necessary and sufficient for optimal association at the septin structure. In this study, we examined the localization of GFP-Bud3 constructs in otherwise wild-type (WT) haploid yeast cells expressing Cdc10-mCherry using fluorescence microscopy; we tested a collection of N- and C-terminal truncations and fusions of separate Bud3 protein elements to identify the smallest domain(s) responsible for bud neck localization. We found that the coordinate action of the central amphipathic helix (residues 847-865) and a partially conserved C-terminal motif (residues 1172-1273) was sufficient to promote bud neck recruitment in the presence of endogenous Bud3. This domain is considerably smaller than the previously characterized C-terminal portion required to physically interact with Bud4 (1221-1636) and utilizes a similar mechanism of pairing membrane association, with a separate localization domain, similar to other non-septin proteins targeted to the division site during cell division.

Reconstructed evolutionary history of the yeast septins Cdc11 and Shs1.

Septins are GTP-binding proteins conserved across metazoans. They can polymerize into extended filaments and, hence, are considered a component of the cytoskeleton. The number of individual septins varies across the tree of life-yeast (Saccharomyces cerevisiae) has seven distinct subunits, a nematode (Caenorhabditis elegans) has two, and humans have 13. However, the overall geometric unit (an apolar hetero-octameric protomer and filaments assembled there from) has been conserved. To understand septin evolutionary variation, we focused on a related pair of yeast subunits (Cdc11 and Shs1) that appear to have arisen from gene duplication within the fungal clade. Either Cdc11 or Shs1 occupies the terminal position within a hetero-octamer, yet Cdc11 is essential for septin function and cell viability, whereas Shs1 is not. To discern the molecular basis of this divergence, we utilized ancestral gene reconstruction to predict, synthesize, and experimentally examine the most recent common ancestor ("Anc.11-S") of Cdc11 and Shs1. Anc.11-S was able to occupy the terminal position within an octamer, just like the modern subunits. Although Anc.11-S supplied many of the known functions of Cdc11, it was unable to replace the distinct function(s) of Shs1. To further evaluate the history of Shs1, additional intermediates along a proposed trajectory from Anc.11-S to yeast Shs1 were generated and tested. We demonstrate that multiple events contributed to the current properties of Shs1: (1) loss of Shs1-Shs1 self-association early after duplication, (2) co-evolution of heterotypic Cdc11-Shs1 interaction between neighboring hetero-octamers, and (3) eventual repurposing and acquisition of novel function(s) for its C-terminal extension domain. Thus, a pair of duplicated proteins, despite constraints imposed by assembly into a highly conserved multi-subunit structure, could evolve new functionality via a complex evolutionary pathway.

Analysis of a Cas12a-based gene-drive system in budding yeast.

The discovery and adaptation of CRISPR/Cas systems within molecular biology has provided advances across biological research, agriculture and human health. Genomic manipulation through use of a CRISPR nuclease and programmed guide RNAs has become a common and widely accessible practice. The identification and introduction of new engineered variants and orthologues of Cas9 as well as alternative CRISPR systems such as the type V group have provided additional molecular options for editing. These include distinct PAM requirements, staggered DNA double-strand break formation, and the ability to multiplex guide RNAs from a single expression construct. Use of CRISPR/Cas has allowed for the construction and testing of a powerful genetic architecture known as a gene drive within eukaryotic model systems. Our previous work developed a drive within budding yeast using Streptococcus pyogenes Cas9. Here, we installed the type V Francisella novicida Cas12a (Cpf1) nuclease gene and its corresponding guide RNA to power a highly efficient artificial gene drive in diploid yeast. We examined the consequence of altering guide length or introduction of individual mutational substitutions to the crRNA sequence. Cas12a-dependent gene-drive function required a guide RNA of at least 18 bp and could not tolerate most changes within the 5' end of the crRNA.

Mathematical modeling of self-contained CRISPR gene drive reversal systems.

There is a critical need for further research into methods to control biological populations. Numerous challenges to agriculture, ecological systems, and human health could be mitigated by the targeted reduction and management of key species (e.g. pests, parasites, and vectors for pathogens). The discovery and adaptation of the CRISPR/Cas editing platform co-opted from bacteria has provided a mechanism for a means to alter an entire population. A CRISPR-based gene drive system can allow for the forced propagation of a genetic element that bypasses Mendelian inheritance which can be used to bias sex determination, install exogenous information, or remove endogenous DNA within an entire species. Laboratory studies have demonstrated the potency by which gene drives can operate within insects and other organisms. However, continued research and eventual application face serious opposition regarding issues of policy, biosafety, effectiveness, and reversal. Previous mathematical work has suggested the use of modified gene drive designs that are limited in spread such as daisy chain or underdominance drives. However, no system has yet been proposed that allows for an inducible reversal mechanism without requiring the introduction of additional individuals. Here, we study gene drive effectiveness, fitness, and inducible drive systems that could respond to external stimuli expanding from a previous frequency-based population model. We find that programmed modification during gene drive propagation could serve as a potent safeguard to either slow or completely reverse drive systems and allow for a return to the original wild-type population.

Modulating CRISPR gene drive activity through nucleocytoplasmic localization of Cas9 in S. cerevisiae.

BACKGROUND: The bacterial CRISPR/Cas genome editing system has provided a major breakthrough in molecular biology. One use of this technology is within a nuclease-based gene drive. This type of system can install a genetic element within a population at unnatural rates. Combatting of vector-borne diseases carried by metazoans could benefit from a delivery system that bypasses traditional Mendelian laws of segregation. Recently, laboratory studies in fungi, insects, and even mice, have demonstrated successful propagation of CRISPR gene drives and the potential utility of this type of mechanism. However, current gene drives still face challenges including evolved resistance, containment, and the consequences of application in wild populations. Additional research into molecular mechanisms that would allow for control, titration, and inhibition of drive systems is needed. RESULTS: In this study, we use artificial gene drives in budding yeast to explore mechanisms to modulate nuclease activity of Cas9 through its nucleocytoplasmic localization. We examine non-native nuclear localization sequences (both NLS and NES) on Cas9 fusion proteins in vivo through fluorescence microscopy and genomic editing. Our results demonstrate that mutational substitutions to nuclear signals and combinatorial fusions can both modulate the level of gene drive activity within a population of cells. CONCLUSIONS: These findings have implications for control of traditional nuclease-dependent editing and use of gene drive systems within other organisms. For instance, initiation of a nuclear export mechanism to Cas9 could serve as a molecular safeguard within an active gene drive to reduce or eliminate editing.

Analysis of CRISPR gene drive design in budding yeast.

Control of biological populations remains a critical goal to address the challenges facing ecosystems and agriculture and those posed by human disease, including pests, parasites, pathogens and invasive species. A particular architecture of the CRISPR/Cas biotechnology - a gene drive - has the potential to modify or eliminate populations on a massive scale. Super-Mendelian inheritance has now been demonstrated in both fungi and metazoans, including disease vectors such as mosquitoes. Studies in yeast and fly model systems have developed a number of molecular safeguards to increase biosafety and control over drive systems in vivo, including titration of nuclease activity, anti-CRISPR-dependent inhibition and use of non-native DNA target sites. We have developed a CRISPR/Cas9 gene drive in Saccharomyces cerevisiae that allows for the safe and rapid examination of alternative drive designs and control mechanisms. In this study, we tested whether non-homologous end-joining (NHEJ) had occurred within diploid cells displaying a loss of the target allele following drive activation and did not detect any instances of NHEJ within multiple sampled populations. We also demonstrated successful multiplexing using two additional non-native target sequences. Furthermore, we extended our analysis of 'resistant' clones that still harboured both the drive and target selection markers following expression of Streptococcus pyogenes Cas9; de novo mutation or NHEJ-based repair could not explain the majority of these heterozygous clones. Finally, we developed a second-generation gene drive in yeast with a guide RNA cassette integrated within the drive locus with a near 100 % success rate; resistant clones in this system could also be reactivated during a second round of Cas9 induction.

Development of a multi-locus CRISPR gene drive system in budding yeast.

The discovery of CRISPR/Cas gene editing has allowed for major advances in many biomedical disciplines and basic research. One arrangement of this biotechnology, a nuclease-based gene drive, can rapidly deliver a genetic element through a given population and studies in fungi and metazoans have demonstrated the success of such a system. This methodology has the potential to control biological populations and contribute to eradication of insect-borne diseases, agricultural pests, and invasive species. However, there remain challenges in the design, optimization, and implementation of gene drives including concerns regarding biosafety, containment, and control/inhibition. Given the numerous gene drive arrangements possible, there is a growing need for more advanced designs. In this study, we use budding yeast to develop an artificial multi-locus gene drive system. Our minimal setup requires only a single copy of S. pyogenes Cas9 and three guide RNAs to propagate three gene drives. We demonstrate how this system could be used for targeted allele replacement of native genes and to suppress NHEJ repair systems by modifying DNA Ligase IV. A multi-locus gene drive configuration provides an expanded suite of options for complex attributes including pathway redundancy, combatting evolved resistance, and safeguards for control, inhibition, or reversal of drive action.

Gene drive inhibition by the anti-CRISPR proteins AcrIIA2 and AcrIIA4 in Saccharomyces cerevisiae.

Given the widespread use and application of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas gene editing system across many fields, a major focus has been the development, engineering and discovery of molecular means to precisely control and regulate the enzymatic function of the Cas9 nuclease. To date, a variety of Cas9 variants and fusion assemblies have been proposed to provide temporally inducible and spatially controlled editing functions. The discovery of a new class of 'anti-CRISPR' proteins, evolved from bacteriophage in response to the prokaryotic nuclease-based immune system, provides a new platform for control over genomic editing. One Cas9-based application of interest to the field of population control is that of the 'gene drive'. Here, we demonstrate use of the AcrIIA2 and AcrIIA4 proteins to inhibit active gene drive systems in budding yeast. Furthermore, an unbiased mutational scan reveals that titration of Cas9 inhibition may be possible by modification of the anti-CRISPR primary sequence.

Some assembly required: Contributions of Tom Stevens' lab to the V-ATPase field.

Tom Stevens' lab has explored the subunit composition and assembly of the yeast V-ATPase for more than 30 years. Early studies helped establish yeast as the predominant model system for study of V-ATPase proton pumps and led to the discovery of protein splicing of the V-ATPase catalytic subunit. The Vma- phenotype, characteristic of loss-of-V-ATPase activity in yeast was key in determining the enzyme's subunit composition via yeast genetics. V-ATPase subunit composition proved to be highly conserved among eukaryotes. Genetic screens for new vma mutants led to identification of a set of dedicated V-ATPase assembly factors and helped unravel the complex pathways for V-ATPase assembly. In later years, exploration of the evolutionary history of several V-ATPase subunits provided new information about the enzyme's structure and function. This review highlights V-ATPase work in the Stevens' lab between 1987 and 2017.

Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae.

Control of biological populations is an ongoing challenge in many fields, including agriculture, biodiversity, ecological preservation, pest control, and the spread of disease. In some cases, such as insects that harbor human pathogens (e.g., malaria), elimination or reduction of a small number of species would have a dramatic impact across the globe. Given the recent discovery and development of the CRISPR-Cas9 gene editing technology, a unique arrangement of this system, a nuclease-based "gene drive," allows for the super-Mendelian spread and forced propagation of a genetic element through a population. Recent studies have demonstrated the ability of a gene drive to rapidly spread within and nearly eliminate insect populations in a laboratory setting. While there are still ongoing technical challenges to design of a more optimal gene drive to be used in wild populations, there are still serious ecological and ethical concerns surrounding the nature of this powerful biological agent. Here, we use budding yeast as a safe and fully contained model system to explore mechanisms that might allow for programmed regulation of gene drive activity. We describe four conserved features of all CRISPR-based drives and demonstrate the ability of each drive component-Cas9 protein level, sgRNA identity, Cas9 nucleocytoplasmic shuttling, and novel Cas9-Cas9 tandem fusions-to modulate drive activity within a population.

Yeast Still a Beast: Diverse Applications of CRISPR/Cas Editing Technology in S. cerevisiae.

The recent discovery and use of CRISPR/Cas9 gene editing technology has provided new opportunities for scientific research in many fields of study including agriculture, genetic disorders, human disease, biotechnology, and basic biological research. The ability to precisely target DNA sequences and either remove, modify, or replace genetic sequences provides a new level of control in nearly all eukaryotic organisms, including budding yeast. Given the many discoveries made in Saccharomyces cerevisiae over the past decades spanning genetics, cell biology, and biochemistry, as well as the development of new technologies that have allowed high throughput screening, robotic automation, and a platform for synthetic genome engineering, the yeast community has also started to recognize the utility and complementary nature of CRISPR-based methodologies. Here we present and review a variety of recent uses of Cas9 in budding yeast-both nuclease dependent and independent applications spanning traditional gene editing and replacement, to transcriptional modulation, to novel uses including the development of living circuitry or robotic platforms for synthetic genome construction. Yeast continues to serve as a powerful model system, yet it can still benefit from use of CRISPR for basic research, industrial application, and innovation of new Cas9-based applications.

Method for Multiplexing CRISPR/Cas9 in Saccharomyces cerevisiae Using Artificial Target DNA Sequences.

Genome manipulation has become more accessible given the advent of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) editing technology. The Cas9 endonuclease binds a single stranded (single guide) RNA (sgRNA) fragment that recruits the complex to a corresponding genomic target sequence where it induces a double stranded break. Eukaryotic repair systems allow for the introduction of exogenous DNA, repair of existing mutations, or deletion of endogenous gene products. Targeting of Cas9 to multiple genomic positions (termed 'multiplexing') is achieved by the expression of multiple sgRNAs within the same nucleus. However, an ongoing concern of the CRISPR field has been the accidental targeting of Cas9 to alternative ('off-target') DNA locations within a genome. We describe the use (dubbed Multiplexing of Cas9 at Artificial Loci) of installed artificial Cas9 target sequences into the yeast genome that allow for (i) multiplexing with a single sgRNA; (ii) a reduction/elimination in possible off-target effects, and (iii) precise control of the placement of the intended target sequence(s).

CRISPR-UnLOCK: Multipurpose Cas9-Based Strategies for Conversion of Yeast Libraries and Strains.

Saccharomyces cerevisiae continues to serve as a powerful model system for both basic biological research and industrial application. The development of genome-wide collections of individually manipulated strains (libraries) has allowed for high-throughput genetic screens and an emerging global view of this single-celled Eukaryote. The success of strain construction has relied on the innate ability of budding yeast to accept foreign DNA and perform homologous recombination, allowing for efficient plasmid construction (in vivo) and integration of desired sequences into the genome. The development of molecular toolkits and "integration cassettes" have provided fungal systems with a collection of strategies for tagging, deleting, or over-expressing target genes; typically, these consist of a C-terminal tag (epitope or fluorescent protein), a universal terminator sequence, and a selectable marker cassette to allow for convenient screening. However, there are logistical and technical obstacles to using these traditional genetic modules for complex strain construction (manipulation of many genomic targets in a single cell) or for the generation of entire genome-wide libraries. The recent introduction of the CRISPR/Cas gene editing technology has provided a powerful methodology for multiplexed editing in many biological systems including yeast. We have developed four distinct uses of the CRISPR biotechnology to generate yeast strains that utilizes the conversion of existing, commonly-used yeast libraries or strains. We present Cas9-based, marker-less methodologies for (i) N-terminal tagging, (ii) C-terminally tagging yeast genes with 18 unique fusions, (iii) conversion of fluorescently-tagged strains into newly engineered (or codon optimized) variants, and finally, (iv) use of a Cas9 "gene drive" system to rapidly achieve a homozygous state for a hypomorphic query allele in a diploid strain. These CRISPR-based methods demonstrate use of targeting universal sequences previously introduced into a genome.

TOR Complex 2-Regulated Protein Kinase Fpk1 Stimulates Endocytosis via Inhibition of Ark1/Prk1-Related Protein Kinase Akl1 in Saccharomyces cerevisiae.

Depending on the stress, plasma membrane alterations activate or inhibit yeast target of rapamycin (TOR) complex 2, which, in turn, upregulates or downregulates the activity of its essential downstream effector, protein kinase Ypk1. Through phosphorylation of multiple substrates, Ypk1 controls many processes that restore homeostasis. One such substrate is protein kinase Fpk1, which is negatively regulated by Ypk1. Fpk1 phosphorylates and stimulates flippases that translocate aminoglycerophospholipids from the outer to the inner leaflet of the plasma membrane. Fpk1 has additional roles, but other substrates were uncharacterized. We show that Fpk1 phosphorylates and inhibits protein kinase Akl1, related to protein kinases Ark1 and Prk1, which modulate the dynamics of actin patch-mediated endocytosis. Akl1 has two Fpk1 phosphorylation sites (Ark1 and Prk1 have none) and is hypophosphorylated when Fpk1 is absent. Conversely, under conditions that inactivate TORC2-Ypk1 signaling, which alleviates Fpk1 inhibition, Akl1 is hyperphosphorylated. Monitoring phosphorylation of known Akl1 substrates (Sla1 and Ent2) confirmed that Akl1 is hyperactive when not phosphorylated by Fpk1. Fpk1-mediated negative regulation of Akl1 enhances endocytosis, because an Akl1 mutant immune to Fpk1 phosphorylation causes faster dissociation of Sla1 from actin patches, confers elevated resistance to doxorubicin (a toxic compound whose entry requires endocytosis), and impedes Lucifer yellow uptake (a marker of fluid phase endocytosis). Thus, TORC2-Ypk1, by regulating Fpk1-mediated phosphorylation of Akl1, adjusts the rate of endocytosis.

Septin-Associated Protein Kinases in the Yeast Saccharomyces cerevisiae.

Septins are a family of eukaryotic GTP-binding proteins that associate into linear rods, which, in turn, polymerize end-on-end into filaments, and further assemble into other, more elaborate super-structures at discrete subcellular locations. Hence, septin-based ensembles are considered elements of the cytoskeleton. One function of these structures that has been well-documented in studies conducted in budding yeast Saccharomyces cerevisiae is to serve as a scaffold that recruits regulatory proteins, which dictate the spatial and temporal control of certain aspects of the cell division cycle. In particular, septin-associated protein kinases couple cell cycle progression with cellular morphogenesis. Thus, septin-containing structures serve as signaling platforms that integrate a multitude of signals and coordinate key downstream networks required for cell cycle passage. This review summarizes what we currently understand about how the action of septin-associated protein kinases and their substrates control information flow to drive the cell cycle into and out of mitosis, to regulate bud growth, and especially to direct timely and efficient execution of cytokinesis and cell abscission. Thus, septin structures represent a regulatory node at the intersection of many signaling pathways. In addition, and importantly, the activities of certain septin-associated protein kinases also regulate the state of organization of the septins themselves, creating a complex feedback loop.

Detection of protein-protein interactions at the septin collar in Saccharomyces cerevisiae using a tripartite split-GFP system.

Various methods can provide a readout of the physical interaction between two biomolecules. A recently described tripartite split-GFP system has the potential to report by direct visualization via a fluorescence signal the intimate association of minimally tagged proteins expressed at their endogenous level in their native cellular milieu and can capture transient or weak interactions. Here we document the utility of this tripartite split-GFP system to assess in living cells protein-protein interactions in a dynamic cytoskeletal structure-the septin collar at the yeast bud neck. We show, first, that for septin-septin interactions, this method yields a robust signal whose strength reflects the known spacing between the subunits in septin filaments and thus serves as a "molecular ruler." Second, the method yields little or no spurious signal even with highly abundant cytosolic proteins readily accessible to the bud neck (including molecular chaperone Hsp82 and glycolytic enzyme Pgk1). Third, using two proteins (Bni5 and Hsl1) that have been shown by other means to bind directly to septins at the bud neck in vivo, we validate that the tripartite split-GFP method yields the same conclusions and further insights about specificity. Finally, we demonstrate the capacity of this approach to uncover additional new information by examining whether three other proteins reported to localize to the bud neck (Nis1, Bud4, and Hof1) are able to interact physically with any of the subunits in the septin collar and, if so, with which ones.

mCAL: A New Approach for Versatile Multiplex Action of Cas9 Using One sgRNA and Loci Flanked by a Programmed Target Sequence.

Genome editing exploiting CRISPR/Cas9 has been adopted widely in academia and in the biotechnology industry to manipulate DNA sequences in diverse organisms. Molecular engineering of Cas9 itself and its guide RNA, and the strategies for using them, have increased efficiency, optimized specificity, reduced inappropriate off-target effects, and introduced modifications for performing other functions (transcriptional regulation, high-resolution imaging, protein recruitment, and high-throughput screening). Moreover, Cas9 has the ability to multiplex, i.e., to act at different genomic targets within the same nucleus. Currently, however, introducing concurrent changes at multiple loci involves: (i) identification of appropriate genomic sites, especially the availability of suitable PAM sequences; (ii) the design, construction, and expression of multiple sgRNA directed against those sites; (iii) potential difficulties in altering essential genes; and (iv) lingering concerns about "off-target" effects. We have devised a new approach that circumvents these drawbacks, as we demonstrate here using the yeast Saccharomyces cerevisiae First, any gene(s) of interest are flanked upstream and downstream with a single unique target sequence that does not normally exist in the genome. Thereafter, expression of one sgRNA and cotransformation with appropriate PCR fragments permits concomitant Cas9-mediated alteration of multiple genes (both essential and nonessential). The system we developed also allows for maintenance of the integrated, inducible Cas9-expression cassette or its simultaneous scarless excision. Our scheme-dubbed mCAL for " M: ultiplexing of C: as9 at A: rtificial L: oci"-can be applied to any organism in which the CRISPR/Cas9 methodology is currently being utilized. In principle, it can be applied to install synthetic sequences into the genome, to generate genomic libraries, and to program strains or cell lines so that they can be conveniently (and repeatedly) manipulated at multiple loci with extremely high efficiency.

Coordinate action of distinct sequence elements localizes checkpoint kinase Hsl1 to the septin collar at the bud neck in Saccharomyces cerevisiae.

Passage through the eukaryotic cell cycle requires processes that are tightly regulated both spatially and temporally. Surveillance mechanisms (checkpoints) exert quality control and impose order on the timing and organization of downstream events by impeding cell cycle progression until the necessary components are available and undamaged and have acted in the proper sequence. In budding yeast, a checkpoint exists that does not allow timely execution of the G2/M transition unless and until a collar of septin filaments has properly assembled at the bud neck, which is the site where subsequent cytokinesis will occur. An essential component of this checkpoint is the large (1518-residue) protein kinase Hsl1, which localizes to the bud neck only if the septin collar has been correctly formed. Hsl1 reportedly interacts with particular septins; however, the precise molecular determinants in Hsl1 responsible for its recruitment to this cellular location during G2 have not been elucidated. We performed a comprehensive mutational dissection and accompanying image analysis to identify the sequence elements within Hsl1 responsible for its localization to the septins at the bud neck. Unexpectedly, we found that this targeting is multipartite. A segment of the central region of Hsl1 (residues 611-950), composed of two tandem, semiredundant but distinct septin-associating elements, is necessary and sufficient for binding to septin filaments both in vitro and in vivo. However, in addition to 611-950, efficient localization of Hsl1 to the septin collar in the cell obligatorily requires generalized targeting to the cytosolic face of the plasma membrane, a function normally provided by the C-terminal phosphatidylserine-binding KA1 domain (residues 1379-1518) in Hsl1 but that can be replaced by other, heterologous phosphatidylserine-binding sequences.

Assembly, molecular organization, and membrane-binding properties of development-specific septins.

Septin complexes display remarkable plasticity in subunit composition, yet how a new subunit assembled into higher-order structures confers different functions is not fully understood. Here, this question is addressed in budding yeast, where during meiosis Spr3 and Spr28 replace the mitotic septin subunits Cdc12 and Cdc11 (and Shs1), respectively. In vitro, the sole stable complex that contains both meiosis-specific septins is a linear Spr28-Spr3-Cdc3-Cdc10-Cdc10-Cdc3-Spr3-Spr28 hetero-octamer. Only coexpressed Spr3 and Spr28 colocalize with Cdc3 and Cdc10 in mitotic cells, indicating that incorporation requires a Spr28-Spr3 protomer. Unlike their mitotic counterparts, Spr28-Spr3-capped rods are unable to form higher-order structures in solution but assemble to form long paired filaments on lipid monolayers containing phosphatidylinositol-4,5-bisphosphate, mimicking presence of this phosphoinositide in the prospore membrane. Spr28 and Spr3 fail to rescue the lethality of a cdc11Δ cdc12Δ mutant, and Cdc11 and Cdc12 fail to restore sporulation proficiency to spr3Δ/spr3Δ spr28Δ/spr28Δ diploids. Thus, specific meiotic and mitotic subunits endow septin complexes with functionally distinct properties.