The RNA-binding protein Puf5 and the HMGB protein Ixr1 contribute to cell cycle progression through the regulation of cell cycle-specific expression of CLB1 in Saccharomyces cerevisiae.

Puf5, a Puf-family RNA-binding protein, binds to 3´ untranslated region of target mRNAs and negatively regulates their expression in Saccharomyces cerevisiae. The puf5Δ mutant shows pleiotropic phenotypes including a weakened cell wall, a temperature-sensitive growth, and a shorter lifespan. To further analyze a role of Puf5 in cell growth, we searched for a multicopy suppressor of the temperature-sensitive growth of the puf5Δ mutant in this study. We found that overexpression of CLB2 encoding B-type cyclin suppressed the temperature-sensitive growth of the puf5Δ mutant. The puf5Δ clb2Δ double mutant displayed a severe growth defect, suggesting that Puf5 positively regulates the expression of a redundant factor with Clb2 in cell cycle progression. We found that expression of CLB1 encoding a redundant B-type cyclin was decreased in the puf5Δ mutant, and that this decrease of the CLB1 expression contributed to the growth defect of the puf5Δ clb2Δ double mutant. Since Puf5 is a negative regulator of the gene expression, we hypothesized that Puf5 negatively regulates the expression of a factor that represses CLB1 expression. We found such a repressor, Ixr1, which is an HMGB (High Mobility Group box B) protein. Deletion of IXR1 restored the decreased expression of CLB1 caused by the puf5Δ mutation and suppressed the growth defect of the puf5Δ clb2Δ double mutant. The expression of IXR1 was negatively regulated by Puf5 in an IXR1 3´ UTR-dependent manner. Our results suggest that IXR1 mRNA is a physiologically important target of Puf5, and that Puf5 and Ixr1 contribute to the cell cycle progression through the regulation of the cell cycle-specific expression of CLB1.

Suppression of Vps13 adaptor protein mutants reveals a central role for PI4P in regulating prospore membrane extension.

Vps13 family proteins are proposed to function in bulk lipid transfer between membranes, but little is known about their regulation. During sporulation of Saccharomyces cerevisiae, Vps13 localizes to the prospore membrane (PSM) via the Spo71-Spo73 adaptor complex. We previously reported that loss of any of these proteins causes PSM extension and subsequent sporulation defects, yet their precise function remains unclear. Here, we performed a genetic screen and identified genes coding for a fragment of phosphatidylinositol (PI) 4-kinase catalytic subunit and PI 4-kinase noncatalytic subunit as multicopy suppressors of spo73Δ. Further genetic and cytological analyses revealed that lowering PI4P levels in the PSM rescues the spo73Δ defects. Furthermore, overexpression of VPS13 and lowering PI4P levels synergistically rescued the defect of a spo71Δ spo73Δ double mutant, suggesting that PI4P might regulate Vps13 function. In addition, we show that an N-terminal fragment of Vps13 has affinity for the endoplasmic reticulum (ER), and ER-plasma membrane (PM) tethers localize along the PSM in a manner dependent on Vps13 and the adaptor complex. These observations suggest that Vps13 and the adaptor complex recruit ER-PM tethers to ER-PSM contact sites. Our analysis revealed that involvement of a phosphoinositide, PI4P, in regulation of Vps13, and also suggest that distinct contact site proteins function cooperatively to promote de novo membrane formation.

Pbp1 mediates the aberrant expression of genes involved in growth defect of ccr4∆ and pop2∆ mutants in yeast Saccharomyces cerevisiae.

CCR4 and POP2 genes encode the catalytic subunit of the Ccr4-Not complex involved in shortening mRNA poly(A) tail in Saccharomyces cerevisiae. The ccr4Δ and pop2∆ mutants exhibit pleiotropic phenotypes such as slow and temperature-sensitive growth, aberrant expression of glucose repression genes and abnormal cell wall synthesis. We previously found that the growth defect of the ccr4Δ and pop2∆ mutants is suppressed by deletion of the PBP1 gene, which encodes poly(A)-binding protein (Pab1)-binding protein 1. In this study, we investigated the functional relationship between Ccr4/Pop2 and Pbp1 by measuring changes in gene expression in ccr4Δ and pop2∆ single mutants and ccr4Δ pbp1∆ and pop2∆ pbp1∆ double mutants. We found that expression of HSP12, HSP26, PIR3, FUS1 and GPH1 was increased in ccr4Δ and pop2∆ single mutants. The pbp1∆ mutation not only restored the growth defect but also reduced the increased expression of those genes found in the ccr4Δ and pop2∆ mutants. Over-expression of PBP1 in the ccr4Δ mutant further increased the expression of HSP12, HSP26, PIR3 and FUS1 and exacerbated the cell growth. These results suggest that the aberrant expression of a subset of genes, which is facilitated by Pbp1, contributes to the pleiotropic phenotypes of the ccr4Δ and pop2∆ mutants.

Spatiotemporal dissection of the trans-Golgi network in budding yeast.

The trans-Golgi network (TGN) acts as a sorting hub for membrane traffic. It receives newly synthesized and recycled proteins, and sorts and delivers them to specific targets such as the plasma membrane, endosomes and lysosomes/vacuoles. Accumulating evidence suggests that the TGN is generated from the trans-most cisterna of the Golgi by maturation, but the detailed transition processes remain obscure. Here, we examine spatiotemporal assembly dynamics of various Golgi/TGN-resident proteins in budding yeast by high-speed and high-resolution spinning-disk confocal microscopy. The Golgi-TGN transition gradually proceeds via at least three successive stages: the 'Golgi stage' where glycosylation occurs; the 'early TGN stage', which receives retrograde traffic; and the 'late TGN stage', where transport carriers are produced. During the stage transition periods, earlier and later markers are often compartmentalized within a cisterna. Furthermore, for the late TGN stage, various types of coat/adaptor proteins exhibit distinct assembly patterns. Taken together, our findings characterize the identity of the TGN as a membrane compartment that is structurally and functionally distinguishable from the Golgi.This article has an associated First Person interview with the first author of the paper.

Pan2-Pan3 complex, together with Ccr4-Not complex, has a role in the cell growth on non-fermentable carbon sources.

There are two major deadenylase complexes, Ccr4-Not and Pan2-Pan3, which shorten the 3' poly(A) tail of mRNA and are conserved from yeast to human. We have previously shown that the Ccr4-mediated deadenylation plays the important role in gene expression regulation in the yeast stationary phase cell. In order to further understand the role of deadenylases in different growth condition, in this study we investigated the effect of deletion of both deadenylases on the cell in non-fermentable carbon containing media. We found that both ccr4Δ and ccr4Δ pan2Δ mutants showed similar growth defect in YPD media: when switched to media containing non-fermentable source (Glycerol-Lactate) only the ccr4Δ grew while the ccr4Δ pan2Δ did not. Ccr4, Pan2, and Pan3 were phosphorylated in GlyLac medium, suggesting that the activities of Ccr4, Pan2, and Pan3 may be regulated by phosphorylation in response to change of carbon sources. To get insights how Ccr4 and Pan2 function in the cell growth in media containing non-fermentable source only, we isolated multicopy suppressors for the growth defect on YPGlyLac media of the ccr4Δ pan2Δ mutant and identified two genes, STM1 and REX2, which encode a ribosome-associated protein and a 3'-5' RNA exonuclease, respectively. Our results suggest that the Pan2-Pan3 complex, together with the Ccr4-Not complex, has important roles in the growth on non-fermentable carbon sources.

The eIF4E-binding protein Eap1 has similar but independent roles in cell growth and gene expression with the cytoplasmic deadenylase Ccr4.

eIF4E-binding proteins (4E-BPs) are translational repressors that compete with eIF4G for binding to eIF4E. Here we investigated the roles of yeast 4E-BPs, Eap1, and Caf20 in cell wall integrity pathway and gene expression. We found that eap1∆ mutation, but not caf20∆ mutation, showed synthetic growth defect with mutation in ROM2 gene encoding Rho1 GEF. The eap1∆ mutation also showed synthetic lethality with mutation in CCR4 gene encoding cytoplasmic deadenylase. Ccr4 functions in the degradation of LRG1 mRNA encoding Rho1 GAP. Eap1-Y109A L114A, which could not bind to eIF4E, did not suppress the synthetic lethality of eap1∆ ccr4∆ mutant, suggesting that 4E-binding of Eap1 is important for its function. We also found that eap1∆ mutant showed the derepression of stress response gene HSP12. 4E-binding of Eap1 was also required for the repression of HSP12 expression. Our results indicate that Eap1 has similar but independent roles in cell growth and gene expression with Ccr4.

Regulation of LRG1 expression by RNA-binding protein Puf5 in the budding yeast cell wall integrity pathway.

The PUF RNA-binding protein Puf5 is involved in regulation of the cell wall integrity (CWI) pathway in yeast. Puf5 negatively regulates expression of LRG1 mRNA, encoding for a GTPase-activating protein for Rho1 small GTPase. Here, we further analyzed the effect of Puf5 on LRG1 expression, together with Ccr4 and Pop2 deadenylases, Dhh1 decapping activator, and other PUF proteins. We found that the growth defect of puf5∆ mutant was enhanced by ccr4∆ mutation, which was partially suppressed by LRG1 deletion. Consistently, Lrg1 protein level was much more up-regulated in ccr4Δ puf5Δ double mutant than in each single mutant. Interestingly, LRG1 poly(A) tail length was longer in ccr4∆ mutant but not in puf5∆ mutant. Thus, Puf5 regulates LRG1 expression independently from Ccr4, although Puf5 recruits the Ccr4-Not deadenylase complex for mRNA destabilization. Unexpectedly, puf6Δ mutation suppressed the growth defect caused by ccr4Δ puf5∆ mutation. Loss of Rpl43a and Rpl43b ribosomal proteins, the previously identified Puf6 interactors, also suppressed the growth defect of ccr4Δ puf5Δ mutant. Our results suggest that Puf5 functions in the CWI pathway by regulating LRG1 expression in a deadenylase-independent manner, and that Puf6 is involved in the Ccr4- and Puf5-mediated regulation of cell growth through association with Rpl43.

Sar1 localizes at the rims of COPII-coated membranes in vivo.

The Sar1 GTPase controls coat assembly on coat protein complex II (COPII)-coated vesicles, which mediate protein transport from the endoplasmic reticulum (ER) to the Golgi. The GTP-bound form of Sar1, activated by the ER-localized guanine nucleotide exchange factor (GEF) Sec12, associates with the ER membrane. GTP hydrolysis by Sar1, stimulated by the COPII-vesicle-localized GTPase-activating protein (GAP) Sec23, in turn causes Sar1 to dissociate from the membrane. Thus, Sar1 is cycled between active and inactive states, and on and off vesicle membranes, but its precise spatiotemporal regulation remains unknown. Here, we examined Sar1 localization on COPII-coated membranes in living Saccharomyces cerevisiae cells. Two-dimensional (2D) observation demonstrated that Sar1 showed modest accumulation around the ER exit sites (ERES) in a manner that was dependent on Sec16 function. Detailed three-dimensional (3D) observation further demonstrated that Sar1 localized at the rims of the COPII-coated membranes, but was excluded from the rest of the COPII membranes. Additionally, a GTP-locked form of Sar1 induced abnormally enlarged COPII-coated structures and covered the entirety of these structures. These results suggested that the reversible membrane association of Sar1 GTPase leads to its localization being restricted to the rims of COPII-coated membranes in vivo.

COPI is essential for Golgi cisternal maturation and dynamics.

Proteins synthesized in the endoplasmic reticulum (ER) are transported to the Golgi and then sorted to their destinations. For their passage through the Golgi, one widely accepted mechanism is cisternal maturation. Cisternal maturation is fulfilled by the retrograde transport of Golgi-resident proteins from later to earlier cisternae, and candidate carriers for this retrograde transport are coat protein complex I (COPI)-coated vesicles. We examined the COPI function in cisternal maturation directly by 4D observation of the transmembrane Golgi-resident proteins in living yeast cells. COPI temperature-sensitive mutants and induced degradation of COPI proteins were used to knockdown COPI function. For both methods, inactivation of COPI subunits Ret1 and Sec21 markedly impaired the transition from cis to medial and to trans cisternae. Furthermore, the movement of cisternae within the cytoplasm was severely restricted when COPI subunits were depleted. Our results demonstrate the essential roles of COPI proteins in retrograde trafficking of the Golgi-resident proteins and dynamics of the Golgi cisternae.

Activation of Rab GTPase Sec4 by its GEF Sec2 is required for prospore membrane formation during sporulation in yeast Saccharomyces cerevisiae.

Sec2 activates Sec4 Rab GTPase as a guanine nucleotide exchange factor for the recruitment of downstream effectors to facilitate tethering and fusion of post-Golgi vesicles at the plasma membrane. During the meiosis and sporulation of budding yeast, post-Golgi vesicles are transported to and fused at the spindle pole body (SPB) to form a de novo membrane, called the prospore membrane. Previous studies have revealed the role of the SPB outer surface called the meiotic outer plaque (MOP) in docking and fusion of post-Golgi vesicles. However, the upstream molecular machinery for post-Golgi vesicular fusion that facilitates prospore membrane formation remains enigmatic. Here, we demonstrate that the GTP exchange factor for Sec4, Sec2, participates in the formation of the prospore membrane. A conditional mutant in which the SEC2 expression is shut off during sporulation showed sporulation defects. Inactivation of Sec2 caused Sec4 targeting defects along the prospore membranes, thereby causing insufficient targeting of downstream effectors and cargo proteins to the prospore membrane. These results suggest that the activation of Sec4 by Sec2 is required for the efficient supply of post-Golgi vesicles to the prospore membrane and thus for prospore membrane formation/extension and subsequent deposition of spore wall materials.

Rab GAP cascade regulates dynamics of Ypt6 in the Golgi traffic.

The Golgi apparatus functions as the central station of membrane traffic in cells, where newly synthesized proteins moving along the secretory pathway merge with proteins recycled from subsequent membrane organelles such as endosomes. A series of Rab GTPases act consecutively and in concert with the maturation of cis- to-trans cisternae of the Golgi apparatus. Rab GTPases control various steps in intracellular membrane traffic by recruiting downstream effector proteins. Here, we report the dynamics of Ypt6, a yeast member of the Rab GTPase family, which mediates the fusion of vesicles from endosomes at the Golgi apparatus. Ypt6 resides temporarily at the Golgi and dissociates into the cytosol upon arrival of Ypt32, another Rab GTPase functioning in the late Golgi. We found that Gyp6, a putative GTPase-activating protein (GAP) for Ypt6, specifically interacts with Ypt32, most likely as an effector. Disruption of GYP6 or introduction of a Rab-GAP activity-deficient mutation in GYP6 resulted in continual residence of Ypt6 at the Golgi. We propose that Ypt32 acts to terminate endosome-to-Golgi traffic through a Rab-GAP cascade as it does for cis-to-trans intra-Golgi traffic. Simultaneous disruption of GAP for early-acting Rab proteins in the Golgi showed appreciable defects in post-Golgi trafficking, but did not significantly affect cell growth.

Phosphatidylinositol 3-kinase and 4-kinase have distinct roles in intracellular trafficking of cellulose synthase complexes in Arabidopsis thaliana.

The oriented deposition of cellulose microfibrils in the plant cell wall plays a crucial role in various plant functions such as cell growth, organ formation and defense responses. Cellulose is synthesized by cellulose synthase complexes (CSCs) embedded in the plasma membrane (PM), which comprise the cellulose synthases (CESAs). The abundance and localization of CSCs at the PM should be strictly controlled for precise regulation of cellulose deposition, which strongly depends on the membrane trafficking system. However, the mechanism of the intracellular transport of CSCs is still poorly understood. In this study, we explored requirements for phosphoinositides (PIs) in CESA trafficking by analyzing the effects of inhibitors of PI synthesis in Arabidopsis thaliana expressing green fluorescent protein-tagged CESA3 (GFP-CESA3). We found that a shift to a sucrose-free condition accelerated re-localization of PM-localized GFP-CESA3 into the periphery of the Golgi apparatus via the clathrin-enriched trans-Golgi network (TGN). Treatment with wortmannin (Wm), an inhibitor of phosphatidylinositol 3- (PI3K) and 4- (PI4K) kinases, and phenylarsine oxide (PAO), a more specific inhibitor for PI4K, inhibited internalization of GFP-CESA3 from the PM. In contrast, treatment with LY294002, which impairs the PI3K activity, did not exert such an inhibitory effect on the sequestration of GFP-CESA3, but caused a predominant accumulation of GFP-CESA3 at the ring-shaped periphery of the Golgi apparatus, resulting in the removal of GFP-CESA3 from the PM. These results indicate that PIs are essential elements for localization and intracellular transport of CESA3 and that PI4K and PI3K are required for distinct steps in secretory and/or endocytic trafficking of CESA3.

The yeast Golgi apparatus.

The Golgi apparatus is an organelle that has been extensively studied in the model eukaryote, yeast. Its morphology varies among yeast species; the Golgi exists as a system of dispersed cisternae in the case of the budding yeast Saccharomyces cerevisiae, whereas the Golgi cisternae in Pichia pastoris and Schizosaccharomyces pombe are organized into stacks. In spite of the different organization, the mechanism of trafficking through the Golgi apparatus is believed to be similar, involving cisternal maturation, in which the resident Golgi proteins are transported backwards while secretory cargo proteins can stay in the cisternae. Questions remain regarding the organization of the yeast Golgi, the regulatory mechanisms that underlie cisternal maturation of the Golgi and transport machinery of cargo proteins through this organelle. Studies using different yeast species have provided hints to these mechanisms.

Dynamic behavior of the trans-golgi network in root tissues of Arabidopsis revealed by super-resolution live imaging.

The trans-Golgi network (TGN) is an important organelle for protein transport at the post-Golgi network, which functions as a sorting station that directs cargo proteins to a variety of destinations including post-Golgi compartments and the extracellular space. However, the functions and dynamics of the TGN in plant cells have not been well understood yet. To elucidate the dynamics of the plant TGN, we established transgenic plants expressing green fluorescent protein (GFP)-SYP43, the ortholog of Tlg2/syntaxin16, which is localized to the TGN in yeast and mammalian cells, under the control of the native promoter as a TGN marker. Observation by confocal laser scanning microscopy and super-resolution confocal live imaging microscopy revealed two types of TGN in Arabidopsis root: the GA-TGNs (Golgi-associated TGNs), located on the trans-side of the Golgi apparatus, and the GI-TGNs (Golgi-released independent TGNs), located away from the Golgi apparatus and behaving independently. The GI-TGNs is derived from a population of GA-TGNs by segregation, although the core of the GA-TGN remains even after the generation of the GI-TGN. We further found that the abundance of the GI-TGNs differs between observed tissues. Our results indicate that the dynamic features of the TGN in plant cells differ from those of animal and yeast cells.

Remodeling of the secretory pathway is coordinated with de novo membrane formation in budding yeast gametogenesis.

Gametogenesis in budding yeast involves a large-scale rearrangement of membrane traffic to allow the de novo formation of a membrane, called the prospore membrane (PSM). However, the mechanism underlying this event is not fully elucidated. Here, we show that the number of endoplasmic reticulum exit sites (ERES) per cell fluctuates and switches from decreasing to increasing upon the onset of PSM formation. Reduction in ERES number, presumably accompanying a transient stall in membrane traffic, resulting in the loss of preexisting Golgi apparatus from the cell, was followed by local ERES regeneration, leading to Golgi reassembly in nascent spores. We have revealed that protein phosphatase-1 (PP-1) and its development-specific subunit, Gip1, promote ERES regeneration through Sec16 foci formation. Furthermore, sed4Δ, a mutant with impaired ERES formation, showed defects in PSM growth and spore formation. Thus, ERES regeneration in nascent spores facilitates the segregation of membrane traffic organelles, leading to PSM growth.

Spatiotemporal dissection of the Golgi apparatus and the ER-Golgi intermediate compartment in budding yeast.

Cargo traffic through the Golgi apparatus is mediated by cisternal maturation, but it remains largely unclear how the cis-cisternae, the earliest Golgi sub-compartment, is generated and how the Golgi matures into the trans-Golgi network (TGN). Here, we use high-speed and high-resolution confocal microscopy to analyze the spatiotemporal dynamics of a diverse set of proteins that reside in and around the Golgi in budding yeast. We find many mobile punctate structures that harbor yeast counterparts of mammalian endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) proteins, which we term 'yeast ERGIC'. It occasionally exhibits approach and contact behavior toward the ER exit sites and gradually matures into the cis-Golgi. Upon treatment with the Golgi-disrupting agent brefeldin A, the ERGIC proteins form larger aggregates corresponding to the Golgi entry core compartment in plants, while cis- and medial-Golgi proteins are absorbed into the ER. We further analyze the dynamics of several late Golgi proteins to better understand the Golgi-TGN transition. Together with our previous studies, we demonstrate a detailed spatiotemporal profile of the entire cisternal maturation process from the ERGIC to the Golgi and further to the TGN.

Automation of yeast spot assays using an affordable liquid handling robot.

The spot assay of the budding yeast Saccharomyces cerevisiae is an experimental method that is used to evaluate the effect of genotypes, medium conditions, and environmental stresses on cell growth and survival. Automation of the spot assay experiments from preparing a dilution series to spotting to observing spots continuously has been implemented based on large laboratory automation devices and robots, especially for high-throughput functional screening assays. However, there has yet to be an affordable solution for the automated spot assays suited to researchers in average laboratories and with high customizability for end-users. To make reproducible spot assay experiments widely available, we have automated the plate-based yeast spot assay of budding yeast using Opentrons OT-2 (OT-2), an affordable liquid-handling robot, and a flatbed scanner. We prepared a 3D-printed mount for the Petri dish to allow for precise placement of the Petri dish inside the OT-2. To account for the uneven height of the agar plates, which were made by human hands, we devised a method to adjust the z-position of the pipette tips based on the weight of each agar plate. During the incubation of the agar plates, a flatbed scanner was used to automatically take images of the agar plates over time, allowing researchers to quantify and compare the cell density within the spots at optimal time points a posteriori. Furthermore, the accuracy of the newly developed automated spot assay was verified by performing spot assays with human experimenters and the OT-2 and quantifying the yeast-grown area of the spots. This study will contribute to the introduction of automated spot assays and the automated acquisition of growth processes in conventional laboratories that are not adapted for high-throughput laboratory automation.

Regulation of CLB6 expression by the cytoplasmic deadenylase Ccr4 through its coding and 3' UTR regions.

RNA stability control contributes to the proper expression of gene products. Messenger RNAs (mRNAs) in eukaryotic cells possess a 5' cap structure and the 3' poly(A) tail which are important for mRNA stability and efficient translation. The Ccr4-Not complex is a major cytoplasmic deadenylase and functions in mRNA degradation. The CLB1-6 genes in Saccharomyces cerevisiae encode B-type cyclins which are involved in the cell cycle progression together with the cyclin-dependent kinase Cdc28. The CLB genes consist of CLB1/2, CLB3/4, and CLB5/6 whose gene products accumulate at the G2-M, S-G2, and late G1 phase, respectively. These Clb protein levels are thought to be mainly regulated by the transcriptional control and the protein stability control. Here we investigated regulation of CLB1-6 expression by Ccr4. Our results show that all CLB1-6 mRNA levels were significantly increased in the ccr4Δ mutant compared to those in wild-type cells. Clb1, Clb4, and Clb6 protein levels were slightly increased in the ccr4Δ mutant, but the Clb2, Clb3, and Clb5 protein levels were similar to those in wild-type cells. Since both CLB6 mRNA and Clb6 protein levels were most significantly increased in the ccr4Δ mutant, we further analyzed the cis-elements for the Ccr4-mediated regulation within CLB6 mRNA. We found that there were destabilizing sequences in both coding sequence and 3' untranslated region (3' UTR). The destabilizing sequences in the coding region were found to be both within and outside the sequences corresponding the cyclin domain. The CLB6 3' UTR was sufficient for mRNA destabilization and decrease of the reporter GFP gene and this destabilization involved Ccr4. Our results suggest that CLB6 expression is regulated by Ccr4 through the coding sequence and 3' UTR of CLB6 mRNA.

Pbp1, the yeast ortholog of human Ataxin-2, functions in the cell growth on non-fermentable carbon sources.

Pbp1, the yeast ortholog of human Ataxin-2, was originally isolated as a poly(A) binding protein (Pab1)-binding protein. Pbp1 regulates the Pan2-Pan3 deadenylase complex, thereby modulating the mRNA stability and translation efficiency. However, the physiological significance of Pbp1 remains unclear since a yeast strain harboring PBP1 deletion grows similarly to wild-type strain on normal glucose-containing medium. In this study, we found that Pbp1 has a role in cell growth on the medium containing non-fermentable carbon sources. While the pbp1Δ mutant showed a similar growth compared to the wild-type cell on a normal glucose-containing medium, the pbp1Δ mutant showed a slower growth on the medium containing glycerol and lactate. Microarray analyses revealed that expressions of the genes involved in gluconeogenesis, such as PCK1 and FBP1, and of the genes involved in mitochondrial function, such as COX10 and COX11, were decreased in the pbp1Δ mutant. Pbp1 regulated the expressions of PCK1 and FBP1 via their promoters, while the expressions of COX10 and COX11 were regulated by Pbp1, not through their promoters. The decreased expressions of COX10 and COX11 in the pbp1Δ mutant were recovered by the loss of Dcp1 decapping enzyme or Xrn1 5'-3'exonuclease. Our results suggest that Pbp1 regulates the expressions of the genes involved in gluconeogenesis and mitochondrial function through multiple mechanisms.

Protein phosphatase type 1-interacting protein Ysw1 is involved in proper septin organization and prospore membrane formation during sporulation.

Sporulation of Saccharomyces cerevisiae is a developmental process in which four haploid spores are generated inside a diploid cell. Gip1, a sporulation-specific targeting subunit of protein phosphatase type 1, together with its catalytic subunit, Glc7, colocalizes with septins along the extending prospore membrane and is required for septin organization and spore wall formation. However, the mechanism by which Gip1-Glc7 phosphatase promotes these events is unclear. We show here that Ysw1, a sporulation-specific coiled-coil protein, has a functional relationship to Gip1-Glc7 phosphatase. Overexpression of YSW1 partially suppresses the sporulation defect of a temperature-sensitive allele of gip1. Ysw1 interacts with Gip1 in a two-hybrid assay, and this interaction is required for suppression. Ysw1 tagged with green fluorescent protein colocalizes with septins and Gip1 along the extending prospore membrane during spore formation. Sporulation is partially defective in ysw1Delta mutant, and cytological analysis revealed that septin structures are perturbed and prospore membrane extension is aberrant in ysw1Delta cells. These results suggest that Ysw1 functions with the Gip1-Glc7 phosphatase to promote proper septin organization and prospore membrane formation.