Loss of Rim15p in shochu yeast alters carbon utilization during barley shochu fermentation

ABSTRACT

Rim15p of the yeast Saccharomyces cerevisiae is a Greatwall-family protein kinase that inhibits alcoholic fermentation during sake brewing. To elucidate the roles of Rim15p in barley shochu fermentation, RIM15 was deleted in shochu yeast. The disruptant did not improve ethanol yield, but altered sugar and glycerol contents in the mash, suggesting that Rim15p has a novel function in carbon utilization.     

Signaling Events of the Rim101 Pathway Occur at the Plasma Membrane in a Ubiquitination-Dependent Manner

In yeast, external alkalization and alteration in plasma membrane lipid asymmetry are sensed by the Rim101 pathway. It is currently under debate whether the signal elicited by external alkalization is transduced to downstream molecules at the plasma membrane or via endocytosis of the Rim21 sensor protein at the late endosome. We found that the downstream molecules, including arrestin-related protein Rim8, calpain-like protein Rim13, and scaffold protein Rim20, accumulated at the plasma membrane upon external alkalization and that the accumulation was dependent on Rim21. Snf7, an endosomal sorting complex required for transport (ESCRT) III subunit also essential for the Rim101 pathway, localized to the plasma membrane, in addition to the late endosome, under alkaline conditions. Snf7 at the plasma membrane but not at the late endosome was shown to be involved in Rim101 signaling. In addition, the Rim101 pathway was normally activated, even when endocytosis was severely impaired. Considering this information as a whole, we propose that Rim101 signaling proceeds at the plasma membrane. We also found that activity of the Rsp5 ubiquitin ligase was required for recruiting the downstream molecules to the plasma membrane, suggesting that ubiquitination mediates Rim101 signaling at the plasma membrane.     

The Ambient pH Response Rim Pathway in Yarrowia lipolytica: Identification of YlRIM9 and Characterization of Its Role in Dimorphism

Yarrowia lipolytica is a dimorphic fungus that secretes either an acidic or an alkaline protease depending on the environmental pH. Previous results have indicated that secretion of the alkaline protease is under control of the pH signaling Pal/Rim pathway originally described in Aspergillus nidulans. Several Y. lipolytica mutants defective in some Rim components of this pathway have been previously isolated and the RIM genes characterized. In the present study, Y. lipolytica RIM9 (palI) gene (YlRIM9) was sequenced from a plasmid (AL414126) of the Genolevures project (the DNA sequence data for YlRIM9 gene has been deposited at EMBL with accession number AJ566902). The derived translation product contains 724 amino acids with a predicted signal peptide and four transmembrane domains in its N-terminal region. We demonstrated that mutation in YlRIM9, as well as in other genes encoding members of the Pal/Rim pathway, did not affect the pH-dependent dimorphic transition of Y. lipolytica. A different pathway must exist in this fungus that controls the effect of pH on dimorphism.     

Pleiotropic functions of the yeast Greatwall-family protein kinase Rim15p: a novel target for the control of alcoholic fermentation

Rim15p, a Greatwall-family protein kinase in yeast Saccharomyces cerevisiae, is required for cellular nutrient responses, such as the entry into quiescence and the induction of meiosis and sporulation. In higher eukaryotes, the orthologous gene products are commonly involved in the cell cycle G₂/M transition. How are these pleiotropic functions generated from a single family of protein kinases? Recent advances in both research fields have identified the conserved Greatwall-mediated signaling pathway and a variety of downstream target molecules. In addition, our studies of S. cerevisiae sake yeast strains revealed that Rim15p also plays a significant role in the control of alcoholic fermentation. Despite an extensive history of research on glycolysis and alcoholic fermentation, there has been no critical clue to artificial modification of fermentation performance of yeast cells. Our finding of an in vivo metabolic regulatory mechanism is expected to provide a major breakthrough in yeast breeding technologies for fermentation applications.     

The endosomal sorting complex required for transport machinery influences haem uptake and capsule elaboration in Cryptococcus neoformans

Iron availability is a key determinant of virulence in the pathogenic fungus Cryptococcus neoformans. Previous work revealed that the ESCRT (endosomal sorting complex required for transport) protein Vps23 functions in iron acquisition, capsule formation and virulence. Here, we further characterized the ESCRT machinery to demonstrate that defects in the ESCRT‐II and III complexes caused reduced capsule attachment, impaired growth on haem and resistance to non‐iron metalloprotoporphyrins. The ESCRT mutants shared several phenotypes with a mutant lacking the pH‐response regulator Rim101, and in other fungi, the ESCRT machinery is known to activate Rim101 via proteolytic cleavage. We therefore expressed a truncated and activated version of Rim101 in the ESCRT mutants and found that this allele restored capsule formation but not growth on haem, thus suggesting a Rim101‐independent contribution to haem uptake. We also demonstrated that the ESCRT machinery acts downstream of the cAMP/protein kinase A pathway to influence capsule elaboration. Defects in the ESCRT components also attenuated virulence in macrophage survival assays and a mouse model of cryptococcosis to a greater extent than reported for loss of Rim101. Overall, these results indicate that the ESCRT complexes function in capsule elaboration, haem uptake and virulence via Rim101‐dependent and independent mechanisms.     

UNC-10在致密核心囊泡分泌过程中的作用

Rim是囊泡分泌活性区中的重要组成蛋白,它与细胞分泌和突触可塑性相关．在秀丽隐感线虫中只存在一种编码Rim的基因即unc-10．我们的研究发现,在线虫中Rim的基因突变unc-10(md1117)会导致致密核心囊泡的分泌缺陷．在活体中,unc-10突变虫系的神经多肽分泌显著下降．此外,在主要分泌致密核心囊泡的ALA神经元内,钙光解释放促发的快相分泌也比野生型减少．运用全内反射荧光显微成像技术,我们观察在unc-10缺失的情况下ALA 神经元中致密核心囊泡的锚定过程,结果显示在细胞膜附近停留的囊泡数目减少,表明囊泡锚定受到阻碍．上述试验结果表明,UNC-10能够影响致密核心囊泡的分泌过程,其机制可能是影响了囊泡的锚定过程．     

Conserved Mode of Interaction between Yeast Bro1 Family V Domains and YP(X)nL Motif-Containing Target Proteins

Yeast Bro1 and Rim20 belong to a family of proteins which possess a common architecture of Bro1 and V domains. Alix and His domain protein tyrosine phosphatase (HD-PTP), mammalian Bro1 family proteins, bind YP(X)nL (n = 1 to 3) motifs in their target proteins through their V domains. In Alix, the Phe residue, which is located in the hydrophobic groove of the V domain, is critical for binding to the YP(X)nL motif. Although the overall sequences are not highly conserved between mammalian and yeast V domains, we show that the conserved Phe residue in the yeast Bro1 V domain is important for binding to its YP(X)nL-containing target protein, Rfu1. Furthermore, we show that Rim20 binds to its target protein Rim101 through the interaction between the V domain of Rim20 and the YPIKL motif of Rim101. The mutation of either the critical Phe residue in the Rim20 V domain or the YPIKL motif of Rim101 affected the Rim20-mediated processing of Rim101. These results suggest that the interactions between V domains and YP(X)nL motif-containing proteins are conserved from yeast to mammalian cells. Moreover, the specificities of each V domain to their target protein suggest that unidentified elements determine the binding specificity.     

Phylogenetic structure and biogeography of the Pacific Rim clade of Sphagnum subgen. Subsecunda: haploid and allodiploid taxa

Although it is an uncommon distribution in seed plants, many bryophytes occur around the Pacific Rim of north‐western North America and eastern Asia. This work focuses on a clade of peatmosses (Sphagnum) that is distributed around the Pacific Rim region, with some individual species found across the total range. The goals were to infer divergent phylogenetic relationships among haploid species in the clade, assess parentage of allopolyploid taxa, and evaluate alternative hypotheses about inter‐ and intraspecific geographical range evolution. Multiple data sets and analyses resolved an ‘Alaska’ clade, distributed across western North America, eastern China and Japan, and an ‘Asia’ clade that includes western Chinese, Thai, Korean, eastern Chinese and Japanese lineages. Allopolyploids have arisen at least four times in the Pacific Rim clade of Sphagnum subgen. Subsecunda; it appears that all allopolyploid origins involved closely related haploid parental taxa. Biogeographical inferences were impacted by topological uncertainty and especially by the biogeographical model utilized to reconstruct ancestral areas. Most analyses converge on the conclusion that the ancestor to this clade of Pacific Rim Sphagnum species was widespread from Alaska south to eastern Asia, but a northern origin for the Alaska subclade was supported by one of the two biogeographical models we employed, under which it was robust to phylogenetic uncertainty.     

Ecological studies of the nemertean fauna in an estuarine system of the northwestern Gulf of Mexico

The distribution and abundance of nemerteans in the brackish-water lakes of Sea Rim State Park, Texas, near the Louisiana border, were studied and compared with other macrobenthos during one year. Six of 93 macrobenthic species collected were nemerteans (0.9% of the total number of specimens). Only one species of nemertean, Carinoma sp., was consistently present. This species is the most ubiquitous and, probably, the most abundant nemertean in the estuarine systems of the Texas coast. Carinoma sp. was collected at Sea Rim from a salinity range of 0–21 ppt and at other Texas estuaries from 2–26 ppt. Preliminary experiments with Carinoma sp. as predator and as prey indicated that it feeds on polychaete worms and in turn is fed upon by white (Penaeus setiferus) and brown (P. aztecus) shrimp.     

Piccolo,a Ca2+ sensor in pancreatic beta-cells. Involvement of cAMP-GEFII.Rim2. Piccolo complex in cAMP-dependent exocytosis

We have previously shown that cAMP-binding protein cAMP-guanidine nucleotide exchange factor II (GEFII) (or Epac2) interacting with Rim2 is involved in cAMP-dependent, protein kinase A-independent exocytosis in pancreatic beta-cells. The action of the cAMP-GEFII.Rim2 complex requires both intracellular cAMP and Ca(2+). Although Rim2 has C(2) domains, its role as a Ca(2+) sensor has remained unclear. In the present investigation, we have discovered that Piccolo, a CAZ (cytoskeletal matrix associated with the active zone) protein in neurons that is structurally related to Rim2, also binds to cAMP-GEFII and that it forms both homodimer and heterodimer with Rim2 in a Ca(2+)-dependent manner, whereas Rim2 alone does not form the homodimer. The association of Piccolo.Rim2 heterodimerization is stronger than Piccolo.Piccolo homodimerization. Treatment of pancreatic islets with antisense oligodeoxynucleotides against Piccolo inhibits insulin secretion induced by cAMP analog 8-bromo-cyclic AMP plus high glucose stimulation. These results suggest that Piccolo serves as a Ca(2+) sensor in exocytosis in pancreatic beta-cells and that the formation of a cAMP-GEFII.Rim2.Piccolo complex is important in cAMP-induced insulin secretion. In addition, this study suggests that CAZ proteins similar to those in neurons are also function in pancreatic beta-cells.