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.

Molecular Phylogeny and Taxonomy of the Coral Genus Cyphastrea (Cnidaria, Scleractinia, Merulinidae) in Japan, With the First Records of Two Species.

The scleractinian coral genus Cyphastrea is widely distributed in the Indo-Pacific region and is common from the subtropical to the warm-temperate regions in Japan. Three new species in this genus have recently been reported from south-eastern Australia or the Red Sea. However, taxonomic and species diversity have been little studied so far in Japan. In this study, we analyzed 112 specimens of Cyphastrea collected from the subtropical to the warm-temperate regions in Japan to clarify the species diversity in the country. This analysis was based on skeletal morphological and molecular analyses using three genetic markers of the nuclear 28S rDNA, histone H3 gene, and the mitochondrial noncoding intergenic region between COI and tRNAmet. The molecular phylogenetic trees showed that our specimens are separated mainly into four clades. Considering the morphological data with the molecular phylogenetic relationships, we confirmed a total of nine species, including two species, C. magna and C. salae, recorded for the first time in Japan. Although eight out of nine species were genetically included within Cyphastrea, one species, C. agassizi, was genetically distant from all other species and was closely related to the genus Leptastrea, suggesting the return of this species to the genus to which it was originally ascribed. Two newly recorded species were reciprocally monophyletic, while the other six species (excluding C. agassizi) clustered in two clades without forming species-specific lineages, including three polyphyletic species. Thus, the species boundary between species in Cyphastrea remains unclear in most species using these three sequenced loci.

LYAR potentiates rRNA synthesis by recruiting BRD2/4 and the MYST-type acetyltransferase KAT7 to rDNA.

Activation of ribosomal RNA (rRNA) synthesis is pivotal during cell growth and proliferation, but its aberrant upregulation may promote tumorigenesis. Here, we demonstrate that the candidate oncoprotein, LYAR, enhances ribosomal DNA (rDNA) transcription. Our data reveal that LYAR binds the histone-associated protein BRD2 without involvement of acetyl-lysine-binding bromodomains and recruits BRD2 to the rDNA promoter and transcribed regions via association with upstream binding factor. We show that BRD2 is required for the recruitment of the MYST-type acetyltransferase KAT7 to rDNA loci, resulting in enhanced local acetylation of histone H4. In addition, LYAR binds a complex of BRD4 and KAT7, which is then recruited to rDNA independently of the BRD2-KAT7 complex to accelerate the local acetylation of both H4 and H3. BRD2 also helps recruit BRD4 to rDNA. By contrast, LYAR has no effect on rDNA methylation or the binding of RNA polymerase I subunits to rDNA. These data suggest that LYAR promotes the association of the BRD2-KAT7 and BRD4-KAT7 complexes with transcription-competent rDNA loci but not to transcriptionally silent rDNA loci, thereby increasing rRNA synthesis by altering the local acetylation status of histone H3 and H4.

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.

ß-1,6-glucan synthesis-associated genes are required for proper spore wall formation in Saccharomyces cerevisiae.

The yeast spore wall is an excellent model to study the assembly of an extracellular macromolecule structure. In the present study, mutants defective in ß-1,6-glucan synthesis, including kre1∆, kre6∆, kre9∆ and big1∆, were sporulated to analyse the effect of ß-1,6-glucan defects on the spore wall. Except for kre6∆, these mutant spores were sensitive to treatment with ether, suggesting that the mutations perturb the integrity of the spore wall. Morphologically, the mutant spores were indistinguishable from wild-type spores. They lacked significant sporulation defects partly because the chitosan layer, which covers the glucan layer, compensated for the damage. The proof for this model was obtained from the effect of the additional deletion of CHS3 that resulted in the absence of the chitosan layer. Among the double mutants, the most severe spore wall deficiency was observed in big1∆ spores. The majority of the big1∆chs3∆ mutants failed to form visible spores at a higher temperature. Given that the big1∆ mutation caused a failure to attach a GPI-anchored reporter, Cwp2-GFP, to the spore wall, ß-1,6-glucan is involved in tethering of GPI-anchored proteins in the spore wall as well as in the vegetative cell wall. Thus, ß-1,6-glucan is required for proper organization of the spore wall. Copyright © 2017 John Wiley & Sons, Ltd.

Applied usage of yeast spores as chitosan beads.

In this study, we present a nonhazardous biological method of producing chitosan beads using the budding yeast Saccharomyces cerevisiae. Yeast cells cultured under conditions of nutritional starvation cease vegetative growth and instead form spores. The spore wall has a multilaminar structure with the chitosan layer as the second outermost layer. Thus, removal of the outermost dityrosine layer by disruption of the DIT1 gene, which is required for dityrosine synthesis, leads to exposure of the chitosan layer at the spore surface. In this way, spores can be made to resemble chitosan beads. Chitosan has adsorptive features and can be used to remove heavy metals and negatively charged molecules from solution. Consistent with this practical application, we find that spores are capable of adsorbing heavy metals such as Cu(2+), Cr(3+), and Cd(2+), and removal of the dityrosine layer further improves the adsorption. Removal of the chitosan layer decreases the adsorption, indicating that chitosan works as an adsorbent in the spores. Besides heavy metals, spores can also adsorb a negatively charged cholesterol derivative, taurocholic acid. Furthermore, chitosan is amenable to chemical modifications, and, consistent with this property, dit1Δ spores can serve as a carrier for immobilization of enzymes. Given that yeast spores are a natural product, our results demonstrate that they, and especially dit1Δ mutants, can be used as chitosan beads and used for multiple purposes.

Use of yeast spores for microencapsulation of enzymes.

Here, we report a novel method to produce microencapsulated enzymes using Saccharomyces cerevisiae spores. In sporulating cells, soluble secreted proteins are transported to the spore wall. Previous work has shown that the spore wall is capable of retaining soluble proteins because its outer layers work as a diffusion barrier. Accordingly, a red fluorescent protein (RFP) fusion of the α-galactosidase, Mel1, expressed in spores was observed in the spore wall even after spores were subjected to a high-salt wash in the presence of detergent. In vegetative cells, however, the cell wall cannot retain the RFP fusion. Although the spore wall prevents diffusion of proteins, it is likely that smaller molecules, such as sugars, pass through it. In fact, spores can contain much higher α-galactosidase activity to digest melibiose than vegetative cells. When present in the spore wall, the enzyme acquires resistance to environmental stresses including enzymatic digestion and high temperatures. The outer layers of the spore wall are required to retain enzymes but also decrease accessibility of the substrates. However, mutants with mild spore wall defects can retain and stabilize the enzyme while still permitting access to the substrate. In addition to Mel1, we also show that spores can retain the invertase. Interestingly the encapsulated invertase has significantly lower activity toward raffinose than toward sucrose.This suggests that substrate selectivity could be altered by the encapsulation.

SPO71 encodes a developmental stage-specific partner for Vps13 in Saccharomyces cerevisiae.

The creation of haploid gametes in yeast, termed spores, requires the de novo formation of membranes within the cytoplasm. These membranes, called prospore membranes, enclose the daughter nuclei generated by meiosis. Proper growth and closure of prospore membranes require the highly conserved Vps13 protein. Mutation of SPO71, a meiosis-specific gene first identified as defective in spore formation, was found to display defects in membrane morphogenesis very similar to those seen in vps13Δ cells. Specifically, prospore membranes are smaller than in the wild type, they fail to close, and membrane vesicles are present within the prospore membrane lumen. As in vps13Δ cells, the levels of phophatidylinositol-4-phosphate are reduced in the prospore membranes of spo71Δ cells. SPO71 is required for the translocation of Vps13 from the endosome to the prospore membrane, and ectopic expression of SPO71 in vegetative cells results in mislocalization of Vps13. Finally, the two proteins can be coprecipitated from sporulating cells. We propose that Spo71 is a sporulation-specific partner for Vps13 and that they act in concert to regulate prospore membrane morphogenesis.

Multifunctional nanobeacon for imaging Thomsen-Friedenreich antigen-associated colorectal cancer.

This research aimed to validate the specificity of the newly developed nanobeacon for imaging the Thomsen-Friedenreich (TF) antigen, a potential biomarker of colorectal cancer. The imaging agent is comprised of a submicron-sized polystyrene nanosphere encapsulated with a Coumarin 6 dye. The surface of the nanosphere was modified with peanut agglutinin (PNA) and poly(N-vinylacetamide (PNVA) moieties. The former binds to Gal-ß(1-3)GalNAc with high affinity while the latter enhances the specificity of PNA for the carbohydrates. The specificity of the nanobeacon was evaluated in human colorectal cancer cells and specimens, and the data were compared with immunohistochemical staining and flow cytometric analysis. Additionally, distribution of the nanobeacon in vivo was assessed using an "intestinal loop" mouse model. Quantitative analysis of the data indicated that approximately 2 µg of PNA were detected for each milligram of the nanobeacon. The nanobeacon specifically reported colorectal tumors by recognizing the tumor-specific antigen through the surface-immobilized PNA. Removal of TF from human colorectal cancer cells and tissues resulted in a loss of fluorescence signal, which suggests the specificity of the probe. Most importantly, the probe was not absorbed systematically in the large intestine upon topical application. As a result, no registered toxicity was associated with the probe. These data demonstrate the potential use of this novel nanobeacon for imaging the TF antigen as a biomarker for the early detection and prediction of the progression of colorectal cancer at the molecular level.

Splicing factor 2-associated protein p32 participates in ribosome biogenesis by regulating the binding of Nop52 and fibrillarin to preribosome particles.

Ribosome biogenesis starts with transcription of the large ribosomal RNA precursor (47S pre-rRNA), which soon combines with numerous factors to form the 90S pre-ribosome in the nucleolus. Although the subsequent separation of the pre-90S particle into pre-40S and pre-60S particles is critical for the production process of mature small and large ribosomal subunits, its molecular mechanisms remain undetermined. Here, we present evidence that p32, fibrillarin (FBL), and Nop52 play key roles in this separation step. Mass-based analyses combined with immunoblotting showed that p32 associated with 155 proteins including 31 rRNA-processing factors (of which nine were components of small subunit processome, and six were those of RIX1 complex), 13 chromatin remodeling components, and six general transcription factors required for RNA polymerase III-mediated transcription. Of these, a late rRNA-processing factor Nop52 interacted directly with p32. Immunocytochemical analyses demonstrated that p32 colocalized with an early rRNA-processing factor FBL or Nop52 in the nucleolus and Cajal bodies, but was excluded from the nucleolus after actinomycin D treatment. p32 was present in the pre-ribosomal fractions prepared by cell fractionation or separated by ultracentrifugation of the nuclear extract. p32 also associated with pre-rRNAs including 47S/45S and 32S pre-rRNAs. Furthermore, knockdown of p32 with a small interfering RNA slowed the early processing from 47S/45S pre-rRNAs to 18S rRNA and 32S pre-rRNA. Finally, Nop52 was found to compete with FBL for binding to p32 probably in the nucleolus. Given the fact that FBL and Nop52 are associated with pre-ribosome particles distinctly different from each other, we suggest that p32 is a new rRNA maturation factor involved in the remodeling from pre-90S particles to pre-40S and pre-60S particles that requires the exchange of FBL for Nop52.

Alg14 organizes the formation of a multiglycosyltransferase complex involved in initiation of lipid-linked oligosaccharide biosynthesis.

Protein N-glycosylation begins with the assembly of a lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER) membrane. The first two steps of LLO biosynthesis are catalyzed by a functional multienzyme complex comprised of the Alg7 GlcNAc phosphotransferase and the heterodimeric Alg13/Alg14 UDP-GlcNAc transferase on the cytosolic face of the ER. In the Alg13/14 glycosyltransferase, Alg14 recruits cytosolic Alg13 to the ER membrane through interaction between their C-termini. Bioinformatic analysis revealed that eukaryotic Alg14 contains an evolved N-terminal region that is missing in bacterial orthologs. Here, we show that this N-terminal region of Saccharomyces cerevisiae Alg14 localize its green fluorescent protein fusion to the ER membrane. Deletion of this region causes defective growth at 38.5°C that can be partially complemented by overexpression of Alg7. Coimmunoprecipitation demonstrated that the N-terminal region of Alg14 is required for direct interaction with Alg7. Our data also show that Alg14 lacking the N-terminal region remains on the ER membrane through a nonperipheral association, suggesting the existence of another membrane-binding site. Mutational studies guided by the 3D structure of Alg14 identified a conserved α-helix involved in the second membrane association site that contributes to an integral interaction and protein stability. We propose a model in which the N- and C-termini of Alg14 coordinate recruitment of catalytic Alg7 and Alg13 to the ER membrane for initiating LLO biosynthesis.

Novel organization of the mitochondrial genome in the deep-sea coral, Madrepora oculata (Hexacorallia, Scleractinia, Oculinidae) and its taxonomic implications.

Madrepora is one of the most ecologically important genera of reef-building scleractinians in the deep sea, occurring from tropical to high-latitude regions. Despite this, the taxonomic affinities and relationships within the genus Madrepora remain unclear. To clarify these issues, we sequenced the mitochondrial (mt) genome of the most widespread Madrepora species, M. oculata, and compared this with data for other scleractinians. The architecture of the M. oculata mt genome was very similar to that of other scleractinians, except for a novel gene rearrangement affecting only cox2 and cox3. This pattern of gene organization was common to four geographically distinct M. oculata individuals as well as the congeneric species M. minutiseptum, but was not shared by other genera that are closely related on the basis of cox1 sequence analysis nor other oculinids, suggesting that it might be unique to Madrepora.

Cell-penetrating peptide-linked polymers as carriers for mucosal vaccine delivery.

We evaluated the potential of poly(N-vinylacetamide-co-acrylic acid) modified with d-octaarginine, which is a typical cell-penetrating peptide, as a carrier for mucosal vaccine delivery. Mice were nasally inoculated four times every seventh day with PBS containing ovalbumin with or without the d-octaarginine-linked polymer. The polymer enhanced the production of ovalbumin-specific immunoglobulin G (IgG) and secreted immunoglobulin A (IgA) in the serum and the nasal cavity, respectively. Ovalbumin internalized into nasal epithelial cells appeared to stimulate IgA production. Ovalbumin transferred to systemic circulation possibly enhanced IgG production. An equivalent dose of the cholera toxin B subunit (CTB), which was used as a positive control, was superior to the polymer in enhancing antibody production; however, dose escalation of the polymer overcame this disadvantage. A similar immunization profile was also observed when ovalbumin was replaced with influenza virus HA vaccines. The polymer induced a vaccine-specific immune response identical to that induced by CTB, irrespective of the antibody type, when its dose was 10 times that of CTB. Our cell-penetrating peptide-linked polymer is a potential candidate for antigen carriers that induce humoral immunity on the mucosal surface and in systemic circulation when nasally coadministered with antigens.

Receptor for advanced glycation end-products (RAGE) mediates phagocytosis in nonprofessional phagocytes.

In mammals, both professional phagocytes and nonprofessional phagocytes (NPPs) can perform phagocytosis. However, limited targets are phagocytosed by NPPs, and thus, the mechanism remains unclear. We find that spores of the yeast Saccharomyces cerevisiae are internalized efficiently by NPPs. Analyses of this phenomenon reveals that RNA fragments derived from cytosolic RNA species are attached to the spore wall, and these fragments serve as ligands to induce spore internalization. Furthermore, we show that a multiligand receptor, RAGE (receptor for advanced glycation end-products), mediates phagocytosis in NPPs. RAGE-mediated phagocytosis is not uniquely induced by spores but is an intrinsic mechanism by which NPPs internalize macromolecules containing RAGE ligands. In fact, artificial particles labeled with polynucleotides, HMGB1, or histone (but not bovine serum albumin) are internalized in NPPs. Our findings provide insight into the molecular basis of phagocytosis by NPPs, a process by which a variety of macromolecules are targeted for internalization.

Identification of novel O-GlcNAc transferase substrates using yeast cells expressing OGT.

O-GlcNAc modification mediated by O-GlcNAc transferase (OGT) is a reversible protein modification in which O-GlcNAc moieties are attached to target proteins in the cytosol, nucleus, and mitochondria. O-GlcNAc moieties attached to proteins can be removed by O-GlcNAcase (OGA). The addition of an O-GlcNAc moiety can influence several aspects of protein function, and aberrant O-GlcNAc modification is linked to a number of diseases. While OGT and OGA are conserved across eukaryotic cells, yeasts lack these enzymes. Previously, we reported that protein O-GlcNAc modification occurred in the budding yeast Saccharomyces cerevisiae when OGT was ectopically expressed. Because yeast cells lack OGA, O-GlcNAc moieties are stably attached to target proteins. Thus, the yeast system may be useful for finding novel OST substrates. By proteomic analysis, we identified 468 O-GlcNAcylated proteins in yeast cells expressing human OGT. Among these proteins, 13 have human orthologues that show more than 30% identity to their corresponding yeast orthologue, and possible glycosylation residues are conserved in these human orthologues. In addition, the orthologues have not been reported as substrates of OGT. We verified that some of these human orthologues are O-GlcNAcylated in cultured human cells. These proteins include an ubiquitin-conjugating enzyme, UBE2D1, and an eRF3-similar protein, HBS1L. Thus, the yeast system would be useful to find previously unknown O-GlcNAcylated proteins and regulatory 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.

Association of human DNA helicase RecQ5beta with RNA polymerase II and its possible role in transcription.

Although RecQ5beta is a ssDNA (single-stranded DNA)-stimulated ATPase and an ATP-dependent DNA helicase with strand-annealing activities, its cellular function remains to be explored. In the present paper, we used immunopurification and MS-based analyses to show that human DNA helicase RecQ5beta is associated with at least four RNAP II (RNA polymerase II) subunits. RecQ5beta was also present in complexes immunoprecipitated using three different antibodies against the large subunit of RNAP II, or in complexes immunoprecipitated using an anti-FLAG antibody against either FLAG-RNAP II 33 kDa subunit or FLAG-Pin1. Different regions of the non-helicase domain of the RecQ5beta molecule were associated with hypophosphorylated and hyperphosphorylated forms of the RNAP II large subunit independently of DNA and RNA. RecQ5beta was also found in nuclear chromatin fractions and associated with the coding regions of the LDL (low-density lipoprotein) receptor and beta-actin genes. Knockdown of the RecQ5beta transcript increased the transcription of those genes. The results of the present study suggest that RecQ5beta has suppressive roles in events associated with RNAP II-dependent transcription.

Septins localize to microtubules during nutritional limitation in Saccharomyces cerevisiae.

BACKGROUND: In Saccharomyces cerevisiae, nutrient limitation stimulates diploid cells to undergo DNA replication and meiosis, followed by the formation of four haploid spores. Septins are a family of proteins that assemble a ring structure at the mother-daughter neck during vegetative growth, where they control cytokinesis. In sporulating cells, the septin ring disassembles and septins relocalize to the prospore membrane. RESULTS: Here, we demonstrate that nutrient limitation triggers a change in the localization of at least two vegetative septins (Cdc10 and Cdc11) from the bud neck to the microtubules. The association of Cdc10 and Cdc11 with microtubules persists into meiosis, and they are found associated with the meiotic spindle until the end of meiosis II. In addition, the meiosis-specific septin Spr28 displays similar behavior, suggesting that this is a common feature of septins. Septin association to microtubules is a consequence of the nutrient limitation signal, since it is also observed when haploid cells are incubated in sporulation medium and when haploid or diploid cells are grown in medium containing non-fermentable carbon sources. Moreover, during meiosis II, when the nascent prospore membrane is formed, septins moved from the microtubules to this membrane. Proper organization of the septins on the membrane requires the sporulation-specific septins Spr3 and Spr28. CONCLUSION: Nutrient limitation in S. cerevisiae triggers the sporulation process, but it also induces the disassembly of the septin bud neck ring and relocalization of the septin subunits to the nucleus. Septins remain associated with microtubules during the meiotic divisions and later, during spore morphogenesis, they are detected associated to the nascent prospore membranes surrounding each nuclear lobe. Septin association to microtubules also occurs during growth in non-fermentable carbon sources.

Poly(N-vinylacetamide) chains enhance lectin-induced biorecognition through the reduction of nonspecific interactions with nontargets.

Lectin-immobilized fluorescent nanospheres were designed with the aim of developing a novel endoscopic imaging agent for the detection of early colorectal cancer. Submicron-sized polystyrene nanospheres with surface poly(N-vinylacetamide) (PNVA) and poly(methacrylic acid) (PMAA) chains encapsulating fluorescein-labeled cholesterol were prepared as a platform of the imaging agent. Peanut agglutinin (PNA) was immobilized on the surface of fluorescent nanospheres through a chemical reaction with PMAA in order to recognize beta-D-galactosyl-(1-3)-N-acetyl-d-galactosamine (Gal-beta(1-3)GalNAc), which is the terminal sugar of the Thomsen-Friedenreich antigen that is specifically expressed on the mucosal side of colorectal cancer cells. The effect of surface structure of nanospheres on the affinity and specificity of immobilized PNA for Gal-beta(1-3)GalNAc was examined. Agglutination of normal and Gal-beta(1-3)GalNAc-expressed erythrocytes in the presence of nanospheres showed that PNA was immobilized actively on the nanosphere surface. Molecular weights of PNVA and PMAA affected the PNA activity most strongly. When the weight-average molecular weight of PNVA was nearly equal to that of PMAA, the affinity of PNA immobilized on the nanosphere surface for Gal-beta(1-3)GalNAc was as strong as that of intact PNA; the specificity for the carbohydrate residue was higher than that of the PNA. Results indicated that PNVA enhanced the specificity of PNA through the reduction of nonspecific interactions between PNA and carbohydrates other than Gal-beta(1-3)GalNAc on the erythrocyte surface without a significant decrease in the affinity.

Schizosaccharomyces pombe Sst4p, a conserved Vps27/Hrs homolog, functions downstream of phosphatidylinositol 3-kinase Pik3p to mediate proper spore formation.

Sporulation of the fission yeast Schizosaccharomyces pombe is a developmental process that generates gametes and that includes the formation of spore envelope precursors called the forespore membranes. Assembly and development of forespore membranes require vesicular trafficking from other intracellular membrane compartments. We have shown that phosphatidylinositol 3-kinase (PtdIns 3-kinase) is required for efficient and proper development of forespore membranes. The role of a FYVE domain protein, Sst4p, a homolog of Vps27p/Hrs, as a downstream factor for PtdIns 3-kinase in sporulation was investigated. sst4Delta asci formed spores with oval-shaped morphology and with reduced viability compared to that of the wild-type spores. The extension of forespore membranes was inefficient, and bubble-like structures emerged from the leading edges of the forespore membranes. Sst4p localization was examined using fluorescent protein fusions and was found to be adjacent to the forespore membranes during sporulation. The localization and function of Sst4p were dependent on its FYVE domain and on PtdIns 3-kinase. Sst4p colocalized and interacted with Hse1p, a homolog of Saccharomyces cerevisiae Hse1p and of mammalian STAM. Mutations in all three UIM domains of the Sst4p/Hse1p complex resulted in formation of spores with abnormal morphology. These results suggest that Sst4p is a downstream factor of PtdIns 3-kinase and functions in forespore membrane formation.