In vivo imaging of fluorescent albumin modified with pyruvylated-human-type complex oligosaccharide reveals sialylation-like biodistribution and kinetics.

Both pyruvylation and sialylation onto the terminus of oligosaccharides of N-glycoproteins seem to be structurally and functionally similar with a property of conferring negative charge. However, detailed molecular characteristics of pyruvylation and sialylation in vivo were elusive. Here, to investigate an effect of terminal pyruvylation to N-glycan on in vivo biodistribution and kinetics, we prepared human serum albumin (HSA) modified with pyruvylated N-glycan (PvG), conjugated with HiLyte Fluor 750 (FL750-PvGHSA). In vivo imaging by using FL750-PvGHSA revealed that terminally pyruvylated N-glycoalbumin was excreted like sialylated N-glycoalbumin, suggesting that pyruvylation mimics sialylation in in vivo biodistribution and kinetics of N-glycoproteins. Terminal pyruvylation onto N-glycans can be a potential tool for a novel glycoengineering strategy.

The endogenous galactofuranosidase GlfH1 hydrolyzes mycobacterial arabinogalactan.

Despite impressive progress made over the past 20 years in our understanding of mycolylarabinogalactan-peptidoglycan (mAGP) biogenesis, the mechanisms by which the tubercle bacillus Mycobacterium tuberculosis adapts its cell wall structure and composition to various environmental conditions, especially during infection, remain poorly understood. Being the central portion of the mAGP complex, arabinogalactan (AG) is believed to be the constituent of the mycobacterial cell envelope that undergoes the least structural changes, but no reports exist supporting this assumption. Herein, using recombinantly expressed mycobacterial protein, bioinformatics analyses, and kinetic and biochemical assays, we demonstrate that the AG can be remodeled by a mycobacterial endogenous enzyme. In particular, we found that the mycobacterial GlfH1 (Rv3096) protein exhibits exo-ß-d-galactofuranose hydrolase activity and is capable of hydrolyzing the galactan chain of AG by recurrent cleavage of the terminal ß-(1,5) and ß-(1,6)-Galf linkages. The characterization of this galactosidase represents a first step toward understanding the remodeling of mycobacterial AG.

Substrate specificities of α1,2- and α1,3-galactosyltransferases and characterization of Gmh1p and Otg1p in Schizosaccharomyces pombe.

In the fission yeast Schizosaccharomyces pombe, α1,2- and α1,3-linked D-galactose (Gal) residues are transferred to N- and O-linked oligosaccharides of glycoproteins by galactosyltransferases. Although the galactomannans are important for cell-cell communication in S. pombe (e.g., in nonsexual aggregation), the mechanisms underlying galactosylation in cells remain unclear. Schizosaccharomyces pombe has 10 galactosyltransferase-related genes: seven belonging to glycosyltransferase (GT) family 34 and three belonging GT family 8. Disruption of all 10 α-galactosyltransferases (strain Δ10GalT) has been shown to result in a complete lack of α-Gal residues. Here, we have investigated the function and substrate specificities of galactosyltransferases in S pombe by using strains expressing single α-galactosyltransferases in the Δ10GalT background. High-performance liquid chromatography (HPLC) analysis of pyridylaminated O-linked oligosaccharides showed that two GT family 34 α1,2-galactosyltransferases (Gma12p and Gmh6p) and two GT family 8 α1,3-galactosyltransferases (Otg2p and Otg3p) are involved in galactosylation of O-linked oligosaccharide. Moreover, 1H-NMR of N-glycans revealed that three GT family 34 α1,2-galactosyltransferases (Gmh1p, Gmh2p and Gmh3p) are required for the galactosylation of N-linked oligosaccharides. Furthermore, HPLC and lectin-blot analysis revealed that Otg1p showed α1,3-galactosyltransferase activity under conditions of co-expression with Gmh6p, indicating that α-1,2-linked galactose is required for the galactosylation activity of Otg1p in S. pombe. In conclusion, eight galactosyltransferases have been shown to have activity in S. pombe with different substrate specificities. These findings will be useful for genetically tailoring the galactosylation of both N- and O-glycans in fission yeast.

Overexpression of cell-wall GPI-anchored proteins restores cell growth of N-glycosylation-defective och1 mutants in Schizosaccharomyces pombe.

The glycoproteins of yeast contain a large outer chain on N-linked oligosaccharides; therefore, yeast is not suitable for producing therapeutic glycoproteins for human use. Using a deletion mutant strain of α1,6-mannosyltransferase (och1Δ), we previously produced humanized N-glycans in fission yeast; however, the Schizosaccharomyces pombe och1Δ cells displayed a growth delay even during vegetative growth, resulting in reduced productivity of heterologous proteins. To overcome this problem, here we performed a genome-wide screen for genes that would suppress the growth defect of temperature-sensitive och1Δ cells. Using a genomic library coupled with screening of 18,000 transformants, we identified two genes (pwp1+, SPBC1E8.05), both encoding GPI-anchored proteins, that increased the growth rate of och1Δ cells, lacking the outer chain. We further showed that a high copy number of the genes was needed to improve the growth rate. Mutational analysis of Pwp1p revealed that the GPI-anchored region of Pwp1p is important in attenuating the growth defect. Analysis of disruptants of pwp1+ and SPBC1E8.05 showed that neither gene was essential for cell viability; however, both mutants were sensitive ß-glucanase, suggesting that Pwp1p and the protein encoded by SPBC1E8.05 non-enzymatically support ß-glucan on the cell-surface of S. pombe. Collectively, our work not only sheds light on the functional relationships between GPI-anchored proteins and N-linked oligosaccharides of glycoproteins in S. pombe, but also supports the application of S. pombe to the production of human glycoprotein. KEY POINTS: • We screened for genes that suppress the growth defect of fission yeast och1Δ cells. • Appropriate expression of GPI-anchored proteins alleviates the growth delay of och1Δ cells. • The GPI-anchor domain of Pwp1p is important for suppressing the growth defect of och1Δ cells.

Structural basis for the specific cleavage of core-fucosylated N-glycans by endo-ß-N-acetylglucosaminidase from the fungus Cordyceps militaris.

N-Linked glycans play important roles in various cellular and immunological events. Endo-ß-N-acetylglucosaminidase (ENGase) can release or transglycosylate N-glycans and is a promising tool for the chemoenzymatic synthesis of glycoproteins with homogeneously modified glycans. The ability of ENGases to act on core-fucosylated glycans is a key factor determining their therapeutic utility because mammalian N-glycans are frequently α-1,6-fucosylated. Although the biochemistries and structures of various ENGases have been studied extensively, the structural basis for the recognition of the core fucose and the asparagine-linked GlcNAc is unclear. Herein, we determined the crystal structures of a core fucose-specific ENGase from the caterpillar fungus Cordyceps militaris (Endo-CoM), which belongs to glycoside hydrolase family 18. Structures complexed with fucose-containing ligands were determined at 1.75-2.35 Å resolutions. The fucose moiety linked to GlcNAc is extensively recognized by protein residues in a round-shaped pocket, whereas the asparagine moiety linked to the GlcNAc is exposed to the solvent. The N-glycan-binding cleft of Endo-CoM is Y-shaped, and several lysine and arginine residues are present at its terminal regions. These structural features were consistent with the activity of Endo-CoM on fucose-containing glycans on rituximab (IgG) and its preference for a sialobiantennary substrate. Comparisons with other ENGases provided structural insights into their core fucose tolerance and specificity. In particular, Endo-F3, a known core fucose-specific ENGase, has a similar fucose-binding pocket, but the surrounding residues are not shared with Endo-CoM. Our study provides a foothold for protein engineering to develop enzymatic tools for the preparation of more effective therapeutic antibodies.

Identification and characterization of a novel, versatile sialidase from a Sphingobacterium that can hydrolyze the glycosides of any sialic acid species at neutral pH.

Bacterial sialidases are widely used to remove sialic acid (Sia) residues from glycans. Most of them cleave the glycosides of N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) under acidic pHs; however, currently available bacterial sialidases had no activity to the glycosides of deaminoneuraminic acid (Kdn). In this study, we found a novel sialidase from Sphingobacterium sp. strain HMA12 that could cleave any of the glycosides of Neu5Ac, Neu5Gc, and Kdn. It also had a broad linkage specificity, i.e., α2,3-, α2,6-, α2,8-, and α2,9-linkages, and the optimal pH at neutral ranges, pH 6.5-7.0. These properties are particularly important when sialidases are applied for in vivo digestion of the cell surface sialosides under physiological conditions. Interestingly, 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (Neu5Ac2en), which is a transition state analog-based inhibitor, competitively inhibited the enzyme-catalyzed reaction for Kdn as well as for Neu5Ac, suggesting that the active site is common to the Neu5Ac and Kdn residues. Taken together, this sialidase is versatile and useful for the in vivo research on sialo-glycoconjugates.

Golgi localization of glycosyltransferases requires Gpp74p in Schizosaccharomyces pombe.

The majority of Golgi glycosyltransferases are type II membrane proteins with a small cytosolic tail at their N-terminus. Several mechanisms for localizing these glycosyltransferases to the Golgi have been proposed. In Saccharomyces cerevisiae, the phosphatidylinositol-4-phosphate-binding protein ScVps74p interacts with the cytosolic tail of a Golgi glycosyltransferase and contributes to its localization. In this study, we investigated whether a similar mechanism functions in the fission yeast Schizosaccharomyces pombe. First, we identified gpp74+ (GPP34 domain-containing Vps74 homolog protein), a gene encoding the S. pombe homolog of S. cerevisiae Vps74p. Deletion of the gpp74+ gene resulted in the missorting of three Golgi glycosyltransferases, SpOch1p, SpMnn9p, and SpOmh1p, to vacuoles, but not SpAnp1p, indicating Gpp74p is required for targeting some glycosyltransferases to the Golgi apparatus. Gpp74p with an N-terminal GFP-tag localized to both the Golgi apparatus and the cytosol. Golgi localization of Gpp74p was dependent on the phosphatidylinositol 4-kinase SpPik1p. Site-directed mutagenesis of hydrophobic and basic amino acids in the cytosolic tails of SpOch1p and SpMnn9p resulted in their missorting to vacuoles, indicating these cytosolic N-terminal residues are important for localization in the Golgi. Unexpectedly, no prominent alternations in protein glycosylation were observed in S. pombe gpp74Δ cells, probably due to the residual Golgi localization of some SpOch1p and SpMnn9p in these cells. Collectively, these results demonstrate that both Gpp74p-dependent and Gpp74p-independent mechanisms are responsible for the Golgi localization of glycosyltransferases to the Golgi in S. pombe. KEY POINTS: • Gpp74p is involved in the localization of glycosyltransferases to the Golgi. • The cytosolic tails of glycosyltransferases are important for Golgi localization. • Gpp74p localizes to the Golgi in a SpPik1p-dependent manner.

Catechol O-methyltransferase homologs in Schizosaccharomyces pombe are response factors to alkaline and salt stress.

How cells of the fission yeast Schizosaccharomyces pombe respond to alkaline stress is not well understood. Here, to elucidate the molecular mechanism underlying the alkaline stress response in S. pombe, we performed DNA microarray analysis. We found that a homolog of human catechol O-methyltransferase 2 (COMT2) is highly upregulated in S. pombe cells exposed to alkaline conditions. We designated the S. pombe homolog as cmt2+ and also identified its paralog, cmt1+, in the S. pombe genome. Reverse transcription PCR confirmed that both cmt1+ and cmt2+ are upregulated within 1 h of exposure to alkaline stress and downregulated within 30 min of returning to an acidic environment. Moreover, we verified that recombinant Cmt proteins exhibit catechol O-methyltransferase activity. To further characterize the expression of cmt1+ and cmt2+, we carried out an EGFP reporter assay using their promoter sequences, which showed that both genes respond not only to alkaline but also to salt stress. Collectively, our findings indicate that the cmt promoter might be an advantageous expression system for use in S. pombe under alkaline culture conditions.

Mutation in fission yeast phosphatidylinositol 4-kinase Pik1 is synthetically lethal with defect in telomere protection protein Pot1.

Fission yeast Pik1p is one of three phosphatidylinositol 4-kinases associated with the Golgi complex, but its function is not fully understood. Deletion of pot1+ causes telomere degradation and chromosome circularization. We searched for the gene which becomes synthetically lethal with pot1Δ. We obtained a novel pik1 mutant, pik1-1, which is synthetically lethal with pot1Δ. We found phosphoinositol 4-phosphate in the Golgi was reduced in pik1-1. To investigate the mechanism of the lethality of the pot1Δ pik1-1 double mutant, we constructed the nmt-pot1-aid pik1-1 strain, where Pot1 function becomes low by drugs, which leads to telomere loss and chromosome circularization, and found pik1-1 mutation does not affect telomere resection and chromosome circularization. Thus, our results suggest that pik1+ is required for the maintenance of circular chromosomes.

Catalytic Activity Profile of Polyphosphate Kinase 1 from Myxococcus xanthus.

Polyphosphate kinase 1 (Ppk1) catalyzes reverse transfer of the terminal phosphate from ATP to form polyphosphate (polyP) and from polyP to form ATP, and is responsible for the synthesis of most of cellular polyPs. When Ppk1 from Myxococcus xanthus was incubated with 0.2 mM polyP60-70 and 1 mM ATP or ADP, the rate of ATP synthesis was approximately 1.5-fold higher than that of polyP synthesis. If in the same reaction the proportion of ADP in the ATP/ADP mixture exceeded one-third, the equilibrium shifted to ATP synthesis, suggesting that M. xanthus Ppk1 preferentially catalyzed ATP formation. At the same time, GTP and GDP were not recognized as substrates by Ppk1. In the absence of polyP, Ppk1 generated ATP and AMP from ADP, and ADP from ATP and AMP, suggesting that the enzyme catalyzed the transfer of a phosphate group between ADP molecules yielding ATP and AMP, thus exhibiting adenylate kinase activity.

GfsA is a ß1,5-galactofuranosyltransferase involved in the biosynthesis of the galactofuran side chain of fungal-type galactomannan in Aspergillus fumigatus.

Previously, we reported that GfsA is a novel galactofuranosyltransferase involved in the biosynthesis of O-glycan, the proper maintenance of fungal morphology, the formation of conidia and anti-fungal resistance in Aspergillus nidulans and A. fumigatus (Komachi Y et al., 2013. GfsA encodes a novel galactofuranosyltransferase involved in biosynthesis of galactofuranose antigen of O-glycan in Aspergillus nidulans and Aspergillus fumigatus. Mol. Microbiol. 90:1054-1073). In the present paper, to gain an in depth-understanding of the enzymatic functions of GfsA in A. fumigatus (AfGfsA), we established an in vitro assay to measure galactofuranosyltransferase activity using purified AfGfsA, UDP-α-d-galactofuranose as a sugar donor, and p-nitrophenyl-ß-d-galactofuranoside as an acceptor substrate. LC/MS, 1H-NMR and methylation analyses of the enzymatic products of AfGfsA revealed that this protein has the ability to transfer galactofuranose to the C-5 position of the ß-galactofuranose residue via a ß-linkage. AfGfsA requires a divalent cation of manganese for maximal activity and consumes UDP-α-d-galactofuranose as a sugar donor. Its optimal pH range is 6.5-7.5 and its optimal temperature range is 20-30°C. 1H-NMR, 13C-NMR and methylation analyses of fungal-type galactomannan extracted from the ∆AfgfsA strain revealed that AfGfsA is responsible for the biosynthesis of ß1,5-galactofuranose in the galactofuran side chain of fungal-type galactomannan. Based on these results, we conclude that AfGfsA acts as a UDP-α-d-galactofuranose: ß-d-galactofuranoside ß1,5-galactofuranosyltransferase in the biosynthetic pathway of galactomannans.

Analysis of an acyl-CoA binding protein in Aspergillus oryzae that undergoes unconventional secretion.

Acyl-CoA binding protein (ACBP) plays important roles in the metabolism of lipids in eukaryotic cells. In the industrially important filamentous fungus Aspergillus oryzae, although we have previously demonstrated that the A. oryzae ACBP (AoACBP) localizes to punctate structures and exhibits long-range motility, which is dependent on autophagy-related proteins, the physiological role of AoACBP remains elusive. Here, we describe identification and characterization of another ACBP from A. oryzae; we named this ACBP as AoAcb2 and accordingly renamed AoACBP as AoAcb1. The deduced amino acid sequence of AoAcb2 lacked a signal peptide. Phylogenetic analysis classified AoAcb2 into a clade that was same as the ACBP Acb1 of the model yeast Saccharomyces cerevisiae, but was different from that of AoAcb1. In contrast to punctate localization of AoAcb1, AoAcb2 was found to be dispersedly distributed in the cytoplasm, as was previously observed for the S. cerevisiae Acb1. Since we could not generate an Aoacb2 disruptant, we created an Aoacb2 conditional mutant that exhibited less growth under Aoacb2-repressed condition, suggesting that Aoacb2 is an essential gene for growth. Moreover, we observed that A. oryzae AoAcb2, but not A. oryzae AoAcb1, was secreted under carbon-starved condition, suggesting that AoAcb2 might be secreted via the unconventional protein secretion (UPS) pathway, just like S. cerevisiae Acb1. We also demonstrated that the unconventional secretion of AoAcb2 was dependent on the t-SNARE AoSso1, but was independent of the autophagy-related protein AoAtg1, suggesting that the unconventional secretion of AoAcb2, unlike that of S. cerevisiae Acb1, via the UPS pathway, is not regulated by the autophagy machinery. Thus, the filamentous fungus A. oryzae harbors two types of ACBPs, one of which appears to be essential for growth and undergoes unconventional secretion.

Characterization of a PA14 domain-containing galactofuranose-specific ß-d-galactofuranosidase from Streptomyces sp.

As a constituent of polysaccharides and glycoconjugates, ß-d-galactofuranose (Galf) exists in several pathogenic microorganisms. Although we recently identified a ß-d-galactofuranosidase (Galf-ase) gene, ORF1110, in the Streptomyces strain JHA19, very little is known about the Galf-ase gene. Here, we characterized a strain, named JHA26, in the culture supernatant of which exhibited Galf-ase activity for 4-nitrophenyl ß-d-galactofuranoside (pNP-ß-d-Galf) as a substrate. Draft genome sequencing of the JHA26 strain revealed a putative gene, termed ORF0643, that encodes Galf-ase containing a PA14 domain, which is thought to function in substrate recognition. The recombinant protein expressed in Escherichia coli showed the Galf-specific Galf-ase activity and also released galactose residue of the polysaccharide galactomannan prepared from Aspergillus fumigatus, suggesting that this enzyme is an exo-type Galf-ase. BLAST searches using the amino acid sequences of ORF0643 and ORF1110 Galf-ases revealed two types of Galf-ases in Actinobacteria, suggesting that Galf-specific Galf-ases may exhibit discrete substrate specificities.

Preparation and biological activities of anti-HER2 monoclonal antibodies with fully core-fucosylated homogeneous bi-antennary complex-type glycans.

Recently, the absence of a core-fucose residue in the N-glycan has been implicated to be important for enhancing antibody-dependent cellular cytotoxicity (ADCC) activity of immunoglobulin G monoclonal antibodies (mAbs). Here, we first prepared anti-HER2 mAbs having two core-fucosylated N-glycan chains with the single G2F, G1aF, G1bF, or G0F structure, together with those having two N-glycan chains with a single non-core-fucosylated corresponding structure for comparison, and determined their biological activities. Dissociation constants of mAbs with core-fucosylated N-glycans bound to recombinant Fcγ-receptor type IIIa variant were 10 times higher than those with the non-core-fucosylated N-glycans, regardless of core glycan structures. mAbs with the core-fucosylated N-glycans had markedly reduced ADCC activities, while those with the non-core-fucosylated N-glycans had high activities. These results indicate that the presence of a core-fucose residue in the N-glycan suppresses the binding to the Fc-receptor and the induction of ADCC of anti-HER2 mAbs.

Highly efficient transglycosylation of sialo-complex-type oligosaccharide using Coprinopsis cinerea endoglycosidase and sugar oxazoline.

OBJECTIVES: To establish an efficient method of chemoenzymatic modification for making N-linked oligosaccharide chains of glycoproteins structurally homogeneous, which crucially affects their bioactivities. RESULTS: Deglycosylated-RNase B (GlcNAc-RNase B; acceptor), sialylglyco (SG)-oxazoline (donor) and an N180H mutant of Coprinopsis cinerea endo-ß-N-acetylglucosaminidase (Endo-CCN180H) were employed. pH 7.5 was ideal for both SG-oxazoline's stability and Endo-CC's transglycosylation reaction. The most efficient reaction conditions for producing glycosylated-RNase B, virtually modified completely with sialo-biantennary-type complex oligosaccharide, were: 80 µg GlcNAc-RNase B, 200 µg SG-oxazoline and 3 µg Endo-CCN180H in 20 µl 20 mM Tris/HCl pH 7.5 at 30 °C for 30-60 min. CONCLUSIONS: This transglycosylation method using SG-oxazoline and Endo-CCN180H is beneficial for producing pharmaceutical glycoproteins modified with homogenous biantennary-complex-type oligosaccharides.

Subcellular localization of acyl-CoA binding protein in Aspergillus oryzae is regulated by autophagy machinery.

In eukaryotic cells, acyl-CoA binding protein (ACBP) is important for cellular activities, such as in lipid metabolism. In the industrially important fungus Aspergillus oryzae, the ACBP, known as AoACBP, has been biochemically characterized, but its physiological function is not known. In the present study, although we could not find any phenotype of AoACBP disruptants in the normal growth conditions, we examined the subcellular localization of AoACBP to understand its physiological function. Using an enhanced green fluorescent protein (EGFP)-tagged AoACBP construct we showed that AoACBP localized to punctate structures in the cytoplasm, some of which moved inside the cells in a microtubule-dependent manner. Further microscopic analyses showed that AoACBP-EGFP co-localized with the autophagy marker protein AoAtg8 tagged with red fluorescent protein (mDsRed). Expression of AoACBP-EGFP in disruptants of autophagy-related genes revealed aggregation of AoACBP-EGFP fluorescence in the cytoplasm of Aoatg1, Aoatg4 and Aoatg8 disruptant cells. However, in cells harboring disruption of Aoatg15, which encodes a lipase for autophagic body, puncta of AoACBP-EGFP fluorescence accumulated in vacuoles, indicating that AoACBP is transported to vacuoles via the autophagy machinery. Collectively, these results suggest the existence of a regulatory mechanism between AoACBP localization and autophagy.

Lysyl-tRNA synthetase from Myxococcus xanthus catalyzes the formation of diadenosine penta- and hexaphosphates from adenosine tetraphosphate.

Myxococcus xanthus lysyl-tRNA synthetase (LysS) produces diadenosine tetraphosphate (Ap4A) from ATP in the presence of Mn(2+); in the present study, it also generated Ap4 from ATP and triphosphate. When ATP and Ap4 were incubated with LysS and pyrophosphatase, first Ap4A, Ap5A, and ADP, and then Ap5, Ap6A, and Ap3A were generated. The results suggest that in the first step, LysS can form lysyl-AMP and lysyl-ADP intermediates from Ap4 and release triphosphate and diphosphate, respectively, whereas in the second step, it can produce Ap5 from lysyl-ADP with triphosphate, and Ap6A from lysyl-ADP with Ap4. In addition, in the presence of Ap4 and pyrophosphatase, but absence of ATP, LysS also generates diadenosine oligophosphates (ApnAs: n = 3-6). These results indicate that LysS has the ability to catalyze the formation of various ApnAs from Ap4 in the presence of pyrophosphatase. Furthermore, the formation of Ap4A by LysS was inhibited by tRNA(Lys) in the presence of 1 mM ATP. To the best of our knowledge, this is the first report of Ap5A and Ap6A synthesis by lysyl-tRNA synthetase.

The amino-terminal hydrophilic region of the vacuolar transporter Avt3p is dispensable for the vacuolar amino acid compartmentalization of Schizosaccharomyces pombe.

Avt3p, a vacuolar amino acid exporter (656 amino acid residues) that is important for vacuolar amino acid compartmentalization as well as spore formation in Schizosaccharomyces pombe, has an extremely long hydrophilic region (approximately 290 amino acid residues) at its N-terminus. Because known functional domains have not been found in this region, its functional role was examined with a deletion mutant avt3(∆1-270) expressed in S. pombe avt3∆ cells. The deletion of this region did not affect its intracellular localization or vacuolar contents of basic amino acids as well as neutral ones. The defect of avt3Δ cells in spore formation was rescued by the expression of avt3+ but was not completely rescued by the expression of avt3(∆1-270). The N-terminal region is thus dispensable for the function of Avt3p as an amino acid exporter, but it is likely to be involved in the role of Avt3p under nutritional starvation conditions.

The fission yeast ß-arrestin-like protein Any1 is involved in TSC-Rheb signaling and the regulation of amino acid transporters.

Rheb GTPase and the Tsc1-Tsc2 protein complex, which serves as a GTPase-activating protein for Rheb, have crucial roles in the regulation of cell growth in response to extracellular conditions. In Schizosaccharomyces pombe, Rheb and Tsc1-Tsc2 regulate cell cycle progression, the onset of meiosis and the uptake of amino acids. In cells lacking Tsc2 (Δtsc2), the amino acid transporter Aat1, which is normally expressed on the plasma membrane under starvation conditions, is confined to the Golgi. Here, we show that the loss of either pub1(+), encoding an E3 ubiquitin ligase, or any1(+), encoding a ß-arrestin-like protein, allows constitutive expression of Aat1 on the plasma membrane in Δtsc2 cells, suggesting that Pub1 and Any1 are required for localization of Aat1 to the Golgi. Subsequent analysis revealed that, in the Golgi, Pub1 and Any1 form a complex that ubiquitylates Aat1. Physical interaction of Pub1 and Any1 is more stable in Δtsc2 cells than in wild-type cells and is independent of Tor2 activity. These results indicate that the TSC-Rheb signaling pathway regulates the localization of amino acid transporters via Pub1 and Any1 in a Tor2-independent manner. Our study demonstrates that, unlike in budding yeast (in which Rsp5 and ARTs, a pair of proteins analogous to Pub1 and Any1, respectively, primarily act to reduce expression of the transporters on plasma membrane when nutrients are abundant), the primary role of fission yeast Pub1 and Any1 is to store the transporter in the Golgi under nutrient-rich conditions.

Vsl1p cooperates with Fsv1p for vacuolar protein transport and homotypic fusion in Schizosaccharomyces pombe.

Members of the SNARE protein family participate in the docking-fusion step of several intracellular vesicular transport events. Saccharomyces cerevisiae Vam7p was identified as a SNARE protein that acts in vacuolar protein transport and membrane fusion. However, in Schizosaccharomyces pombe, there have been no reports regarding the counterpart of Vam7p. Here, we found that, although the SPCC594.06c gene has low similarity to Vam7p, the product of SPCC594.06c has a PX domain and SNARE motif like Vam7p, and thus we designated the gene Sch. pombe vsl1(+) (Vam7-like protein 1). The vsl1Δ cells showed no obvious defect in vacuolar protein transport. However, cells of the vsl1Δ mutant with a deletion of fsv1(+), which encodes another SNARE protein, displayed extreme defects in vacuolar protein transport and vacuolar morphology. Vsl1p was localized to the vacuolar membrane and prevacuolar compartment, and its PX domain was essential for proper localization. Expression of the fusion protein GFP-Vsl1p was able to suppress ZnCl2 sensitivity and the vacuolar protein sorting defect in the fsv1Δ cells. Moreover, GFP-Vsl1p was mislocalized in a pep12Δ mutant and in cells overexpressing fsv1(+). Importantly, overexpression of Sac. cerevisiae VAM7 could suppress the sensitivity to ZnCl2 of vsl1Δ cells and the vacuolar morphology defect of vsl1Δfsv1Δ cells in Sch. pombe. Taken together, these data suggest that Vsl1p and Fsv1p are required for vacuolar protein transport and membrane fusion, and they function cooperatively with Pep12p in the same membrane-trafficking step.