Ssu72 phosphatase deficiency leads to spindle crossing during the second meiotic division process.

Ssu72 is a component of the yeast cleavage/polyadenylation factor (CPF) complex, which catalyzes the dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II at S5-P and S7-P. It has been shown that Ssu72 phosphatase is involved in regulating chromosome cohesion during mitosis. To further clarify whether Ssu72 phosphatase affects chromosome separation during meiotic division in Schizosaccharomyces pombe, we utilized green fluorescent protein (GFP) to label centromeres and red fluorescent protein to label microtubule protein Atb2. The entire meiotic chromosome separation process of ssu72∆ cells was observed in real-time under fluorescence microscope. It was found that two spindles of ssu72∆ cells crossed during the metaphase and anaphase of the second meiotic division, and this spindle crossing led to a new type of spore defect distribution pattern. The results of this study can provide important reference significance for studying the roles of phosphatase Ssu72 in higher organisms.

Expression of an endo-rhamnogalacturonase from Aspergillus aculeatus enhances release of Arabidopsis transparent mucilage.

Mucilage is a gelatinous and sticky hydrophilic polysaccharide released from epidermal cells of seed coat after the hydration of mature seeds and is composed primarily of unbranched rhamnogalacturonan I (RG-I). In this study, we produced a recombinant endo-RG-I hydrolase from Aspergillus aculeatus (AaRhgA) in the fission yeast Schizosaccharomyces pombe and examined its substrate preference for pyridylaminated (PA) RG-I with the various degrees of polymerization (DP). Recombinant AaRhgA requires PA-RG-I with a DP of 10 or higher for its hydrolase activity. We heterologously expressed the AarhgA gene under the strong constitutive promoter, cauliflower mosaic virus 35S promoter, in Arabidopsis thaliana. In a series of biochemical analyses of each mucilage fraction released from the water-imbibed seeds of the transgenic plants, we found the enhanced deposition of the transparent mucilage layer that existed in the peripheral regions of the adherent mucilage and was not stained with ruthenium red. This study demonstrated the feasibility of manipulating the mucilage organization by heterologous expression of the endo-RG-I hydrolase.

Live-cell fluorescence imaging and optogenetic control of PKA kinase activity in fission yeast Schizosaccharomyces pombe.

The cAMP-PKA signaling pathway plays a crucial role in sensing and responding to nutrient availability in the fission yeast Schizosaccharomyces pombe. This pathway monitors external glucose levels to control cell growth and sexual differentiation. However, the temporal dynamics of the cAMP-PKA pathway in response to external stimuli remains unclear mainly due to the lack of tools to quantitatively visualize the activity of the pathway. Here, we report the development of the kinase translocation reporter (KTR)-based biosensor spPKA-KTR1.0, which allows us to measure the dynamics of PKA activity in fission yeast cells. The spPKA-KTR1.0 is derived from the transcription factor Rst2, which translocates from the nucleus to the cytoplasm upon PKA activation. We found that spPKA-KTR1.0 translocates between the nucleus and cytoplasm in a cAMP-PKA pathway-dependent manner, indicating that the spPKA-KTR1.0 is a reliable indicator of the PKA activity in fission yeast cells. In addition, we implemented a system that simultaneously visualizes and manipulates the cAMP-PKA signaling dynamics by introducing bPAC, a photoactivatable adenylate cyclase, in combination with spPKA-KTR1.0. This system offers an opportunity for investigating the role of the signaling dynamics of the cAMP-PKA pathway in fission yeast cells with higher temporal resolution.

Phosphoregulation of DNA repair via the Rad51 auxiliary factor Swi5-Sfr1.

Homologous recombination (HR) is a major pathway for the repair of DNA double-strand breaks, the most severe form of DNA damage. The Rad51 protein is central to HR, but multiple auxiliary factors regulate its activity. The heterodimeric Swi5-Sfr1 complex is one such factor. It was previously shown that two sites within the intrinsically disordered domain of Sfr1 are critical for the interaction with Rad51. Here, we show that phosphorylation of five residues within this domain regulates the interaction of Swi5-Sfr1 with Rad51. Biochemical reconstitutions demonstrated that a phosphomimetic mutant version of Swi5-Sfr1 is defective in both the physical and functional interaction with Rad51. This translated to a defect in DNA repair, with the phosphomimetic mutant yeast strain phenocopying a previously established interaction mutant. Interestingly, a strain in which Sfr1 phosphorylation was blocked also displayed sensitivity to DNA damage. Taken together, we propose that controlled phosphorylation of Sfr1 is important for the role of Swi5-Sfr1 in promoting Rad51-dependent DNA repair.

Identification of novel coenzyme Q10 biosynthetic proteins Coq11 and Coq12 in Schizosaccharomyces pombe.

Coenzyme Q (CoQ) is an essential component of the electron transport system in aerobic organisms. CoQ10 has ten isoprene units in its quinone structure and is especially valuable as a food supplement. However, the CoQ biosynthetic pathway has not been fully elucidated, including synthesis of the p-hydroxybenzoic acid (PHB) precursor to form a quinone backbone. To identify the novel components of CoQ10 synthesis, we investigated CoQ10 production in 400 Schizosaccharomyces pombe gene-deleted strains in which individual mitochondrial proteins were lost. We found that deletion of coq11 (an S. cerevisiae COQ11 homolog) and a novel gene designated coq12 lowered CoQ levels to ∼4% of that of the WT strain. Addition of PHB or p-hydroxybenzaldehyde restored the CoQ content and growth and lowered hydrogen sulfide production of the Δcoq12 strain, but these compounds did not affect the Δcoq11 strain. The primary structure of Coq12 has a flavin reductase motif coupled with an NAD+ reductase domain. We determined that purified Coq12 protein from S. pombe displayed NAD+ reductase activity when incubated with ethanol-extracted substrate of S. pombe. Because purified Coq12 from Escherichia coli did not exhibit reductase activity under the same conditions, an extra protein is thought to be necessary for its activity. Analysis of Coq12-interacting proteins by LC-MS/MS revealed interactions with other Coq proteins, suggesting formation of a complex. Thus, our analysis indicates that Coq12 is required for PHB synthesis, and it has diverged among species.

Identification of a small RhoA GTPase inhibitor effective in fission yeast and human cells.

The Rho GTPase family proteins are key regulators of cytoskeletal dynamics. Deregulated activity of Rho GTPases is associated with cancers and neurodegenerative diseases, and their potential as drug targets has long been recognized. Using an economically effective drug screening workflow in fission yeast and human cells, we have identified a Rho GTPase inhibitor, O1. By a suppressor mutant screen in fission yeast, we find a point mutation in the rho1 gene that confers resistance to O1. Consistent with the idea that O1 is the direct inhibitor of Rho1, O1 reduced the cellular amount of activated, GTP-bound Rho1 in wild-type cells, but not in the O1-resistant mutant cells, in which the evolutionarily conserved Ala62 residue is mutated to Thr. Similarly, O1 inhibits activity of the human orthologue RhoA GTPase in tissue culture cells. Our studies illustrate the power of yeast phenotypic screens in the identification and characterization of drugs relevant to human cells and have identified a novel GTPase inhibitor for fission yeast and human cells.

Fission Yeast PUF Proteins Puf3 and Puf4 Are Novel Regulators of PI4P5K Signaling.

Phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) is a highly conserved enzyme that generates phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) by phosphorylating phosphatidylinositol 4-phosphate (PI(4)P). Schizosaccharomyces pombe (S. pombe) its3-1 is a loss-of-function mutation in the essential its3+ gene that encodes a PI4P5K. Its3 regulates cell proliferation, cytokinesis, cell integrity, and membrane trafficking, but little is known about the regulatory mechanisms of Its3. To identify regulators of Its3, we performed a genetic screening utilizing the high-temperature sensitivity (TS) of its3-1 and identified puf3+ and puf4+, encoding Pumilio/PUF family RNA-binding proteins as multicopy suppressors of its3-1 cells. The deletions of the PUF domains in the puf3+ and puf4+ genes resulted in the reduced ability to suppress its3-1, suggesting that the suppression by Puf3 and Puf4 may involve their RNA-binding activities. The gene knockout of Puf4, but not that of Puf3, exacerbated the TS of its3-1. Interestingly, mutant Its3 expression levels both at mRNA and protein levels were lower than those of the wild-type (WT) Its3. Consistently, the overexpression of the mutant its3-1 gene suppressed the its3-1 phenotypes. Notably, Puf3 and Puf4 overexpression increased the mRNA and protein expression levels of both Its3 and Its3-1. Collectively, our genetic screening revealed a functional relationship between the Pumilio/PUF family RNA-binding proteins and PI4P5K.

Experimental and Computational Studies on the Biotransformation of Pseudopyronines with Human Cytochrome P450 CYP4F2.

The secondary metabolite pseudopyronine B, isolated from Pseudomonas mosselii P33, was biotransformed by human P450 enzymes, heterologously expressed in the fission yeast Schizosaccharomyces pombe. Small-scale studies confirmed that both CYP4F2 and CYP4F3A were capable of oxidizing the substrate, with the former achieving a higher yield. In larger-scale studies using CYP4F2, three new oxidation products were obtained, the structures of which were elucidated by UV-vis, 1D and 2D NMR, and HR-MS spectroscopy. These corresponded to hydroxylated, carboxylated, and ester derivatives (1-3) of pseudopyronine B, all of which had been oxidized exclusively at the ω-position of the C-6 alkyl chain. In silico homology modeling experiments highlighted key interactions between oxygen atoms of the pyrone ring and two serine residues and a histidine residue of CYP4F2, which hold the substrate in a suitable orientation for oxidation at the terminus of the C-6 alkyl chain. Additional modeling studies with all three pseudopyronines revealed that the seven-carbon alkyl chain of pseudopyronine B was the perfect length for oxidation, with the terminal carbon lying close to the heme iron. The antibacterial activity of the substrates and three oxidation products was also assessed, revealing that oxidation at the ω-position removes all antimicrobial activity. This study both increases the range of known substrates for human CYF4F2 and CYP4F3A enzymes and demonstrates their utility in producing additional natural product derivatives.

Cohesin ATPase activities regulate DNA binding and coiled-coil configuration.

The cohesin complex is required for sister chromatid cohesion and genome compaction. Cohesin coiled coils (CCs) can fold at break sites near midpoints to bring head and hinge domains, located at opposite ends of coiled coils, into proximity. Whether ATPase activities in the head play a role in this conformational change is yet to be known. Here, we dissected functions of cohesin ATPase activities in cohesin dynamics in Schizosaccharomyces pombe. Isolation and characterization of cohesin ATPase temperature-sensitive (ts) mutants indicate that both ATPase domains are required for proper chromosome segregation. Unbiased screening of spontaneous suppressor mutations rescuing the temperature lethality of cohesin ATPase mutants identified several suppressor hotspots in cohesin that located outside of ATPase domains. Then, we performed comprehensive saturation mutagenesis targeted to these suppressor hotspots. Large numbers of the identified suppressor mutations indicated several different ways to compensate for the ATPase mutants: 1) Substitutions to amino acids with smaller side chains in coiled coils at break sites around midpoints may enable folding and extension of coiled coils more easily; 2) substitutions to arginine in the DNA binding region of the head may enhance DNA binding; or 3) substitutions to hydrophobic amino acids in coiled coils, connecting the head and interacting with other subunits, may alter conformation of coiled coils close to the head. These results reflect serial structural changes in cohesin driven by its ATPase activities potentially for packaging DNAs.

Core control principles of the eukaryotic cell cycle.

Cyclin-dependent kinases (CDKs) lie at the heart of eukaryotic cell cycle control, with different cyclin-CDK complexes initiating DNA replication (S-CDKs) and mitosis (M-CDKs)1,2. However, the principles on which cyclin-CDK complexes organize the temporal order of cell cycle events are contentious3. One model proposes that S-CDKs and M-CDKs are functionally specialized, with substantially different substrate specificities to execute different cell cycle events4-6. A second model proposes that S-CDKs and M-CDKs are redundant with each other, with both acting as sources of overall CDK activity7,8. In this model, increasing CDK activity, rather than CDK substrate specificity, orders cell cycle events9,10. Here we reconcile these two views of core cell cycle control. Using phosphoproteomic assays of in vivo CDK activity in fission yeast, we find that S-CDK and M-CDK substrate specificities are remarkably similar, showing that S-CDKs and M-CDKs are not completely specialized for S phase and mitosis alone. Normally, S-CDK cannot drive mitosis but can do so when protein phosphatase 1 is removed from the centrosome. Thus, increasing S-CDK activity in vivo is sufficient to overcome substrate specificity differences between S-CDK and M-CDK, and allows S-CDK to carry out M-CDK function. Therefore, we unite the two opposing views of cell cycle control, showing that the core cell cycle engine is largely based on a quantitative increase in CDK activity through the cell cycle, combined with minor and surmountable qualitative differences in catalytic specialization of S-CDKs and M-CDKs.

Fission yeast Duf89 and Duf8901 are cobalt/nickel-dependent phosphatase-pyrophosphatases that act via a covalent aspartyl-phosphate intermediate.

Domain of Unknown Function 89 (DUF89) proteins are metal-dependent phosphohydrolases. Exemplary DUF89 enzymes differ in their metal and phosphosubstrate preferences. Here, we interrogated the activities and structures of two DUF89 paralogs from fission yeast-Duf89 and Duf8901. We find that Duf89 and Duf8901 are cobalt/nickel-dependent phosphohydrolases adept at hydrolyzing p-nitrophenylphosphate and PPi. Crystal structures of metal-free Duf89 and Co2+-bound Duf8901 disclosed two enzyme conformations that differed with respect to the position of a three-helix module, which is either oriented away from the active site in Duf89 or forms a lid over the active site in Duf8901. Lid closure results in a 16 Å movement of Duf8901 Asp195, vis-à-vis Asp199 in Duf89, that brings Asp195 into contact with an octahedrally coordinated cobalt. Reaction of Duf8901 with BeCl2 and NaF in the presence of divalent cations Co2+, Ni2+, or Zn2+ generated covalent Duf8901-(Asp248)-beryllium trifluoride (BeF3)•Co2+, Duf8901-(Asp248)-BeF3•Ni2+, or Duf8901-(Asp248)-BeF3•Zn2+ adducts, the structures of which suggest a two-step catalytic mechanism via formation and hydrolysis of an enzyme-(aspartyl)-phosphate intermediate. Alanine mutations of Duf8901 Asp248, Asn249, Lys401, Asp286, and Asp195 that interact with BeF3•Co2+ squelched p-nitrophenylphosphatase activity. A 1.8 Å structure of a Duf8901-(Asp248)-AlF4-OH2•Co2+ transition-state mimetic suggests an associative mechanism in which Asp195 and Asp363 orient and activate the water nucleophile. Whereas deletion of the duf89 gene elicited a phenotype in which expression of phosphate homeostasis gene pho1 was derepressed, deleting duf8901 did not, thereby hinting that the DUF89 paralogs have distinct functional repertoires in vivo.

Stress-dependent inhibition of polarized cell growth through unbalancing the GEF/GAP regulation of Cdc42.

Cdc42 GTPase rules cell polarity and growth in fission yeast. It is negatively and positively regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), respectively. Active Cdc42-GTP localizes to the poles, where it associates with numerous proteins constituting the polarity module. However, little is known about its downregulation. We describe here that oxidative stress causes Sty1-kinase-dependent Cdc42 inactivation at cell poles. Both the amount of active Cdc42 at tips and cell length inversely correlate with Sty1 activity, explaining the elongated morphology of Δsty1 cells. We have created stress-blinded cell poles either by eliminating two Cdc42 GAPs or through the constitutive tethering of Gef1 to cell tips, and we biochemically demonstrate that the GAPs Rga3/6 and the GEF Gef1 are direct substrates of Sty1. We propose that phosphorylation of Rga3/6 and Gef1 mediates the Sty1-dependent inhibition of Cdc42 at cell tips, halting polarized growth during stress adaptation.

The Hsp90 cochaperone TTT promotes cotranslational maturation of PIKKs prior to complex assembly.

Phosphatidylinositol 3-kinase-related kinases (PIKKs) are a family of kinases that control fundamental processes, including cell growth, DNA damage repair, and gene expression. Although their regulation and activities are well characterized, little is known about how PIKKs fold and assemble into active complexes. Previous work has identified a heat shock protein 90 (Hsp90) cochaperone, the TTT complex, that specifically stabilizes PIKKs. Here, we describe a mechanism by which TTT promotes their de novo maturation in fission yeast. We show that TTT recognizes newly synthesized PIKKs during translation. Although PIKKs form multimeric complexes, we find that they do not engage in cotranslational assembly with their partners. Rather, our findings suggest a model by which TTT protects nascent PIKK polypeptides from misfolding and degradation because PIKKs acquire their native state after translation is terminated. Thus, PIKK maturation and assembly are temporally segregated, suggesting that the biogenesis of large complexes requires both dedicated chaperones and cotranslational interactions between subunits.

Non-mitochondrial aconitase regulates the expression of iron-uptake genes by controlling the RNA turnover process in fission yeast.

Aconitase, a highly conserved protein across all domains of life, functions in converting citrate to isocitrate in the tricarboxylic acid cycle. Cytosolic aconitase is also known to act as an iron regulatory protein in mammals, binding to the RNA hairpin structures known as iron-responsive elements within the untranslated regions of specific RNAs. Aconitase-2 (Aco2) in fission yeast is a fusion protein consisting of an aconitase and a mitochondrial ribosomal protein, bL21, residing not only in mitochondria but also in cytosol and the nucleus. To investigate the role of Aco2 in the nucleus and cytoplasm of fission yeast, we analyzed the transcriptome of aco2ΔN mutant that is deleted of nuclear localization signal (NLS). RNA sequencing revealed that the aco2ΔN mutation caused increase in mRNAs encoding iron uptake transporters, such as Str1, Str3, and Shu1. The half-lives of mRNAs for these genes were found to be significantly longer in the aco2ΔN mutant than the wild-type strain, suggesting the role of Aco2 in mRNA turnover. The three conserved cysteines required for the catalytic activity of aconitase were not necessary for this role. The UV cross-linking RNA immunoprecipitation analysis revealed that Aco2 directly bound to the mRNAs of iron uptake transporters. Aco2-mediated degradation of iron-uptake mRNAs appears to utilize exoribonuclease pathway that involves Rrp6 as evidenced by genetic interactions. These results reveal a novel role of non-mitochondrial aconitase protein in the mRNA turnover in fission yeast to fine-tune iron homeostasis, independent of regulation by transcriptional repressor Fep1.

Nickel induced cell impairments are negatively regulated by the Tor1 kinase in Schizosaccharomyces pombe.

In our study we investigated the effect of different nickel (NiSO4·6H2O) (Ni) concentrations on cell division, cellular morphology and ionome homeostasis of the eukaryotic model organism Schizosaccharomyces pombe. Target of rapamycin (TOR) protein kinase is one of the key regulators of cell growth under different environmental stresses. We analyzed the effect of Ni on cell strains lacking the Tor1 signaling pathway utilizing light-absorbance spectroscopy, visualization, microscopy and inductively coupled plasma optical emission spectroscopy. Interestingly, our findings revealed that Ni mediated cell growth alterations are noticeably lower in Tor1 deficient cells. Greater size of Tor1 depleted cells reached similar quantitative parameters to wild type cells upon incubation with 400 µM Ni. Differences of ion levels among the two tested yeast strains were detected even before Ni addition. Addition of high concentration (1 mM) of the heavy metal, representing acute contamination, caused considerable changes in the ionome of both strains. Strikingly, Tor1 deficient cells displayed largely reduced Ni content after treatment compared to wild type controls (644.1 ± 49 vs. 2096.8 ± 75 µg/g), suggesting its significant role in Ni trafficking. Together our results predict yet undefined role for the Tor1 signaling in metal uptake and/or metabolism.

Synthesis of modified nucleotide polymers by the poly(U) polymerase Cid1: application to direct RNA sequencing on nanopores.

Understanding transcriptomes requires documenting the structures, modifications, and abundances of RNAs as well as their proximity to other molecules. The methods that make this possible depend critically on enzymes (including mutant derivatives) that act on nucleic acids for capturing and sequencing RNA. We tested two 3' nucleotidyl transferases, Saccharomyces cerevisiae poly(A) polymerase and Schizosaccharomyces pombe Cid1, for the ability to add base and sugar modified rNTPs to free RNA 3' ends, eventually focusing on Cid1. Although unable to polymerize ΨTP or 1meΨTP, Cid1 can use 5meUTP and 4thioUTP. Surprisingly, Cid1 can use inosine triphosphate to add poly(I) to the 3' ends of a wide variety of RNA molecules. Most poly(A) mRNAs efficiently acquire a uniform tract of about 50 inosine residues from Cid1, whereas non-poly(A) RNAs acquire longer, more heterogeneous tails. Here we test these activities for use in direct RNA sequencing on nanopores, and find that Cid1-mediated poly(I)-tailing permits detection and quantification of both mRNAs and non-poly(A) RNAs simultaneously, as well as enabling the analysis of nascent RNAs associated with RNA polymerase II. Poly(I) produces a different current trace than poly(A), enabling recognition of native RNA 3' end sequence lost by in vitro poly(A) addition. Addition of poly(I) by Cid1 offers a broadly useful alternative to poly(A) capture for direct RNA sequencing on nanopores.

The Multiple Functions of Rho GTPases in Fission Yeasts.

The Rho family of GTPases represents highly conserved molecular switches involved in a plethora of physiological processes. Fission yeast Schizosaccharomyces pombe has become a fundamental model organism to study the functions of Rho GTPases over the past few decades. In recent years, another fission yeast species, Schizosaccharomyces japonicus, has come into focus offering insight into evolutionary changes within the genus. Both fission yeasts contain only six Rho-type GTPases that are spatiotemporally controlled by multiple guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), and whose intricate regulation in response to external cues is starting to be uncovered. In the present review, we will outline and discuss the current knowledge and recent advances on how the fission yeasts Rho family GTPases regulate essential physiological processes such as morphogenesis and polarity, cellular integrity, cytokinesis and cellular differentiation.

Substrate Phosphorylation Rates as an In Vivo Measurement of Kinase Activity.

Measuring kinase activity in different in vivo contexts is crucial for understanding the mechanism and functions of protein kinases, such as the cyclin-dependent kinases (Cdks) and other cell cycle kinases. Here, I present the rationale and the experimental framework for an alternative approach to measure kinase activity that is based on estimating substrate phosphorylation rates in vivo. The approach presented was first developed for experiments performed to measure Cdk1 activity at different stages of the fission yeast S. pombe's cell cycle [Swaffer et al., Cell 167:1750-1761, 2016]. However, it also affords a more generalizable framework that can be adaptable to other systems and kinases, as long as specific, rapid, and reversible kinase inhibition is possible. Briefly this involves transient and reversible kinase inhibition to dephosphorylate kinase substrates in vivo, followed by quantitative measurements of phosphorylation after inhibition is removed.

Expression of Mug14 is regulated by the transcription factor Rst2 through the cAMP-dependent protein kinase pathway in Schizosaccharomyces pombe.

The cAMP-dependent protein kinase (Pka1) regulates many cellular events, including sexual development and glycogenesis, and response to the limitation of glucose, in Schizosaccharomyces pombe. Despite its importance in many cellular events, the targets of the cAMP/PKA pathway have not been fully investigated. Here, we demonstrate that the expression of mug14 is induced by downregulation of the cAMP/PKA pathway and limitation of glucose. This regulation is dependent on the function of Rst2, a transcription factor that regulates transition from mitosis to meiosis. The loss of the C2H2-type zinc finger domain in Rst2, termed Rst2 (C2H2∆), abolished the induction of Mug14 expression. Upon deletion of the stress starvation response element of the S. pombe (STREP: CCCCTC) sequence, which is a potential binding site of Rst2 on mug14, in the pka1∆ strain, its induction was abolished. The expression of Mug14 was significantly reduced and delayed by the limitation of glucose and also by nitrogen starvation in the rst2∆ strain. Mug14 is known to share a common function with Mde1 and Mta3 in the methionine salvage pathway, but the expression of mde1 and mta3 mRNAs was not enhanced by pka1 deletion and limitation of glucose. We conclude that the expression of Mug14 is upregulated by Rst2 under the control of the cAMP/PKA signaling pathway, which senses the limitation of glucose.

Crystal structure of the Thr316Ala mutant of a yeast JAMM deubiquitinase: implication of active-site loop dynamics in catalysis.

AMSH, an endosome-associated deubiquitinase (DUB) with a high specificity for Lys63-linked polyubiquitin chains, plays an important role in endosomal-lysosomal sorting and down-regulation of cell-surface receptors. AMSH belongs to the JAMM family of DUBs that contain two insertion segments, Ins-1 and Ins-2, in the catalytic domain relative to the JAMM core found in the archaebacterial AfJAMM. Structural analyses of the AMSH homologs human AMSH-LP and fission yeast Sst2 reveal a flap-like structure formed by Ins-2 near the active site that appears to open and close during its catalytic cycle. A conserved phenylalanine residue of the flap interacts with a conserved aspartate residue of the Ins-1 ß-turn to form a closed `lid' over the active site in the substrate-bound state. Analyses of these two residues (Phe403 and Asp315) in Sst2 showed that their interaction plays an important role in controlling the flexibility of Ins-2. The Lys63-linked diubiquitin substrate-bound form of Sst2 showed that the conserved phenylalanine also interacts with Thr316 of Ins-1, which is substituted by tyrosine in other AMSH orthologs. Although Thr316 makes no direct interaction with the substrate, its mutation to alanine resulted in a significant loss of activity. In order to understand the contribution of Thr316 to catalysis, the crystal structure of this mutant was determined. In spite of the effect of the mutation on catalytic activity, the structure of the Sst2 Thr316Ala mutant did not reveal significant changes in either the overall structure or the active-site arrangement relative to the wild type. The Phe403-Thr316 van der Waals interaction is impaired by the Thr316Ala mutation, abrogating the adoption of the closed active-site conformation required for catalysis. Since van der Waals interactions with phenylalanine are conserved across substrate-bound forms of AMSH-LP and Sst2, these interactions may be critical for loop immobilization and the positioning of the isopeptide bond of Lys63-linked polyubiquitin-chain substrates.