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Telomerase is a ribonucleoprotein enzyme, which maintains genome integrity in eukaryotes and ensures continuous cellular proliferation. Telomerase holoenzyme from the thermotolerant yeast Hansenula polymorpha, in addition to the catalytic subunit (TERT) and telomerase RNA (TER), contains accessory proteins Est1 and Est3, which are essential for in vivo telomerase function. Here we report the high-resolution structure of Est3 from Hansenula polymorpha (HpEst3) in solution, as well as the characterization of its functional relationships with other components of telomerase. The overall structure of HpEst3 is similar to that of Est3 from Saccharomyces cerevisiae and human TPP1. We have shown that telomerase activity in H. polymorpha relies on both Est3 and Est1 proteins in a functionally symmetrical manner. The absence of either Est3 or Est1 prevents formation of a stable ribonucleoprotein complex, weakens binding of a second protein to TER, and decreases the amount of cellular TERT, presumably due to the destabilization of telomerase RNP. NMR probing has shown no direct in vitro interactions of free Est3 either with the N-terminal domain of TERT or with DNA or RNA fragments mimicking the probable telomerase environment. Our findings corroborate the idea that telomerase possesses the evolutionarily variable functionality within the conservative structural context.

Cell division cycle 5 (Cdc5) is a highly conserved nucleic acid binding protein among eukaryotes and plays critical roles in development. Cdc5 can simultaneously bind to DNA and RNA by its N-terminal DNA-binding domain (DBD), but molecular mechanisms describing its nucleic acid recognition and the regulation of development through its nucleic acid binding remain unclear. Herein, we present a crystal structure of the N-terminal DBD of MoCdc5 (MoCdc5-DBD) from the rice blast fungus Magnaporthe oryzae. Residue K100 of MoCdc5 is on the periphery of a positively charged groove that is formed by K42, K45, R47, and N92 and is evolutionally conserved. Mutation of K100 significantly reduces the affinity of MoCdc5-DBD to a Cdc5-binding element but not to a conventional myeloblastosis (Myb) domain-binding element, suggesting that K100 is a key residue of the high binding affinity to Cdc5-binding element. Another conserved residue (R31) is located close to the U6 RNA in the structure of the spliceosome, and its mutation dramatically reduces the binding capacity of MoCdc5-DBD for U6 RNA. Importantly, mutations in these key residues, including R31, K42, and K100 in AtCDC5, an Arabidopsis thaliana ortholog of MoCdc5, greatly impair the functions of AtCDC5, resulting in pleiotropic development defects and reduced levels of primary microRNA transcripts. Taken together, our findings suggest that Cdc5-DBD binds nucleic acids with two distinct binding surfaces, one for DNA and another for RNA, which together contribute to establishing the regulation mechanism of Cdc5 on development through nucleic acid binding., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Kinetochores are the multiprotein complexes that link chromosomal centromeres to mitotic-spindle microtubules. Budding yeast centromeres comprise three sequential "centromere-determining elements", CDEI, II, and III. CDEI (8 bp) and CDEIII (∼25 bp) are conserved between Kluyveromyces lactis and Saccharomyces cerevisiae, but CDEII in the former is twice as long (160 bp) as CDEII in the latter (80 bp). The CBF3 complex recognizes CDEIII and is required for assembly of a centromeric nucleosome, which in turn recruits other kinetochore components. To understand differences in centromeric nucleosome assembly between K. lactis and S. cerevisiae, we determined the structure of a K. lactis CBF3 complex by electron cryomicroscopy at ∼4 Å resolution and compared it with published structures of S. cerevisiae CBF3. We show differences in the pose of Ndc10 and discuss potential models of the K. lactis centromeric nucleosome that account for the extended CDEII length., (Copyright © 2019. Published by Elsevier Ltd.)

ABC transporter proteins are involved in active transport, both in prokaryotes and eukaryotes. Sequence analysis of nucleotide binding domains (NBDs) of ABC proteins from all taxa revealed a well-conserved new motif having the signature: xT/ShxE/DNhxF, located between Q-loop and ABC signature sequence. A recent structure of an ABC transporter, ABCG5/G8 highlighted the motif as an essential structural determinant of inter-domain crosstalk and termed it as E-helix. We carried out an extensive computational analysis to unravel important structural entities alongside E-helix which plausibly play role in the interlocking mechanism of NBD with TMD. We identified E-helix to be a central structural moiety which interacts with three helices and an intracellular loop that leads to the transmembrane domain. Considering its wide occurrence, we examined the importance of this motif in one representative multidrug ABC transporter of Candida albicans, Cdr1p. The motif residues were replaced by alanines both individually as well as in combinations. The GFP-tagged versions of mutant proteins were overexpressed in Saccharomyces cerevisiae. Overall, our mutational data suggested that this motif plays a role in the maintenance of proper structural fold and/or inter-domain contacts in Cdr1p. We, thus, unveil an essential structural motif in ABC superfamily transporters., (Copyright © 2019. Published by Elsevier B.V.)

The SET1/MLL family of histone methyltransferases is conserved in eukaryotes and regulates transcription by catalyzing histone H3K4 mono-, di-, and tri-methylation. These enzymes form a common five-subunit catalytic core whose assembly is critical for their basal and regulated enzymatic activities through unknown mechanisms. Here, we present the crystal structure of the intact yeast COMPASS histone methyltransferase catalytic module consisting of Swd1, Swd3, Bre2, Sdc1, and Set1. The complex is organized by Swd1, whose conserved C-terminal tail not only nucleates Swd3 and a Bre2-Sdc1 subcomplex, but also joins Set1 to construct a regulatory pocket next to the catalytic site. This inter-subunit pocket is targeted by a previously unrecognized enzyme-modulating motif in Swd3 and features a doorstop-style mechanism dictating substrate selectivity among SET1/MLL family members. By spatially mapping the functional components of COMPASS, our results provide a structural framework for understanding the multifaceted functions and regulation of the H3K4 methyltransferase family., (Published by Elsevier Inc.)

The interaction of the eukaryotic initiation factor 4G (eIF4G) with the cap-binding protein eIF4E initiates cap-dependent translation and is regulated by the 4E-binding proteins (4E-BPs), which compete with eIF4G to repress translation. Metazoan eIF4G and 4E-BPs interact with eIF4E via canonical and non-canonical motifs that bind to the dorsal and lateral surface of eIF4E in a bipartite recognition mode. However, previous studies pointed to mechanistic differences in how fungi and metazoans regulate protein synthesis. We present crystal structures of the yeast eIF4E bound to two yeast 4E-BPs, p20 and Eap1p, as well as crystal structures of a fungal eIF4E-eIF4G complex. We demonstrate that the core principles of molecular recognition of eIF4E are in fact highly conserved among translational activators and repressors in eukaryotes. Finally, we reveal that highly specialized structural motifs do exist and serve to modulate the affinity of protein-protein interactions that regulate cap-dependent translation initiation in fungi.

Peroxisomes are a major cellular compartment of eukaryotic cells, and are involved in a variety of metabolic functions and pathways according to species, cell type and environmental conditions. Their biogenesis relies on conserved genes known as PEX genes that encode peroxin proteins. Peroxisomal membrane proteins and peroxisomal matrix proteins are generated in the cytosol and are subsequently imported into the peroxisome post-translationally. Matrix proteins containing a peroxisomal targeting signal type 1 (PTS1) are recognized by the cycling receptor Pex5p and transported to the peroxisomal lumen. Pex5p docking, release of the cargo into the lumen and recycling involve a number of peroxins, but a key player is the Pex4p-Pex22p complex described in this manuscript. Pex4p from the yeast Saccharomyces cerevisiae is a ubiquitin-conjugating enzyme that is anchored on the cytosolic side of the peroxisomal membrane through its binding partner Pex22p, which acts as both a docking site and a co-activator of Pex4p. As Pex5p undergoes recycling and release, the Pex4p-Pex22p complex is essential for monoubiquitination at the conserved cysteine residue of Pex5p. The absence of Pex4p-Pex22p inhibits Pex5p recycling and hence PTS1 protein import. This article reports the crystallization of Pex4p and of the Pex4p-Pex22p complex from the yeast Hansenula polymorpha, and data collection from their crystals to 2.0 and 2.85 Å resolution, respectively. The resulting structures are likely to provide important insights to understand the molecular mechanism of the Pex4p-Pex22p complex and its role in peroxisome biogenesis.

SUMO proteases of the SENP/Ulp family are master regulators of both sumoylation and desumoylation and regulate SUMO homeostasis in eukaryotic cells. SUMO conjugates rapidly increase in response to cellular stress, including nutrient starvation, hypoxia, osmotic stress, DNA damage, heat shock, and other proteotoxic stressors. Nevertheless, little is known about the regulation and targeting of SUMO proteases during stress. To this end we have undertaken a detailed comparison of the SUMO-binding activity of the budding yeast protein Ulp1 (ScUlp1) and its ortholog in the thermotolerant yeast Kluyveromyces marxianus, KmUlp1. We find that the catalytic UD domains of both ScUlp1 and KmUlp1 show a high degree of sequence conservation, complement a ulp1Δ mutant in vivo, and process a SUMO precursor in vitro. Next, to compare the SUMO-trapping features of both SUMO proteases we produced catalytically inactive recombinant fragments of the UD domains of ScUlp1 and KmUlp1, termed ScUTAG and KmUTAG respectively. Both ScUTAG and KmUTAG were able to efficiently bind a variety of purified SUMO isoforms and bound immobilized SUMO1 with nanomolar affinity. However, KmUTAG showed a greatly enhanced ability to bind SUMO and SUMO-modified proteins in the presence of oxidative, temperature and other stressors that induce protein misfolding. We also investigated whether a SUMO-interacting motif (SIM) in the UD domain of KmULP1 that is not conserved in ScUlp1 may contribute to the SUMO-binding properties of KmUTAG. In summary, our data reveal important details about how SUMO proteases target and bind their sumoylated substrates, especially under stress conditions. We also show that the robust pan-SUMO binding features of KmUTAG can be exploited to detect and study SUMO-modified proteins in cell culture systems.

Proteasomes are responsible for protein turnover in eukaryotic cells, degrading short-lived species but also removing improperly folded or oxidatively damaged ones. Dysfunction of a proteasome results in gradual accumulation of misfolded/damaged proteins, leading to their aggregation. It has been postulated that proteasome activators may facilitate removal of such aggregation-prone proteins and thus prevent development of neurodegenerative disorders. However, the discovery of pharmacologically relevant compounds is hindered by insufficient structural understanding of the activation process. In this study we provide a model peptidic activator of human proteasome and analyze the structure-activity relationship within this novel scaffold. The binding mode of the activator at the relevant pocket within the proteasome has been determined by X-ray crystallography. This crystal structure provides an important basis for rational design of pharmacological compounds. Moreover, by providing a novel insight into the proteasome gating mechanism, our results allow the commonly accepted model of proteasome regulation to be revisited.

Hsp104 is a yeast member of the Hsp100 family which functions as a molecular chaperone to disaggregate misfolded polypeptides. To understand the mechanism by which the Hsp104 N-terminal domain (NTD) interacts with its peptide substrates, crystal structures of the Hsp104 NTDs from Saccharomyces cerevisiae (ScHsp104NTD) and Candida albicans (CaHsp104NTD) have been determined at high resolution. The structures of ScHsp104NTD and CaHsp104NTD reveal that the yeast Hsp104 NTD may utilize a conserved putative peptide-binding groove to interact with misfolded polypeptides. In the crystal structures ScHsp104NTD forms a homodimer, while CaHsp104NTD exists as a monomer. The consecutive residues Gln105, Gln106 and Lys107, and Lys141 around the putative peptide-binding groove mediate the monomer-monomer interactions within the ScHsp104NTD homodimer. Dimer formation by ScHsp104NTD suggests that the Hsp104 NTD may specifically interact with polyQ regions of prion-prone proteins. The data may reveal the mechanism by which Hsp104 NTD functions to suppress and/or dissolve prions.

Peroxisomes proliferate by growth and division of pre-existing peroxisomes or could arise de novo. Though the de novo pathway of peroxisome biogenesis is a more recent discovery, several studies have highlighted key mechanistic details of the pathway. The endoplasmic reticulum (ER) is the primary source of lipids and proteins for the newly-formed peroxisomes. More recently, an intricate sorting process functioning at the ER has been proposed, that segregates specific PMPs first to peroxisome-specific ER domains (pER) and then assembles PMPs selectively into distinct pre-peroxisomal vesicles (ppVs) that later fuse to form import-competent peroxisomes. In addition, plausible roles of the three key peroxins Pex3, Pex16 and Pex19, which are also central to the growth and division pathway, have been suggested in the de novo process. In this review, we discuss key developments and highlight the unexplored avenues in de novo peroxisome biogenesis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Polysaccharide endotransglycosylases (PETs) are the cell wall-modifying enzymes of fungi and plants. They catalyze random endo-splitting of the polysaccharide donor molecule and transfer of the newly formed reducing sugar residue to the nonreducing end of an acceptor molecule which can be a polysaccharide or an oligosaccharide. Owing to their important role in the cell wall formation, the inhibition of PETs represents an attractive strategy in the fight against fungal infections. We have elaborated two variants of a versatile high-throughput microplate fluorimetric assay that could be used for effective identification of PETs and screening of their inhibitors. Both assays use the respective polysaccharides as the donors and sulforhodamine-labeled oligosaccharides as the acceptors but differ from each other by mode of how the labeled polysaccharide products of transglycosylation are separated from the unreacted oligosaccharide acceptors. In the first variant, the reactions take place in a layer of agar gel laid on the bottoms of the wells of a microtitration plate. After the reaction, the high-Mr transglycosylation products are precipitated with 66 % ethanol and retained within the gel while the low-Mr products and the unreacted acceptors are washed out. In the second variant, the donor polysaccharides are adsorbed to the surface of a microplate well and remain adsorbed there also after becoming labeled in the course of the transglycosylation reaction whereas the unused low-Mr acceptors are washed out. As a proof of versatility, assays of heterologously expressed transglycosylases ScGas1, ScCrh1, and ScCrh2 from the yeast Saccharomyces cerevisiae, CaPhr1 and CaPhr2 from Candida albicans, and of a plant xyloglucan endotransglycosylase (XET) are demonstrated.

Eukaryotic ribosome biogenesis depends on several hundred assembly factors to produce functional 40S and 60S ribosomal subunits. The final phase of 60S subunit biogenesis is cytoplasmic maturation, which includes the proofreading of functional centers of the 60S subunit and the release of several ribosome biogenesis factors. We report the cryo-electron microscopy (cryo-EM) structure of the yeast 60S subunit in complex with the biogenesis factors Rei1, Arx1, and Alb1 at 3.4 Å resolution. In addition to the network of interactions formed by Alb1, the structure reveals a mechanism for ensuring the integrity of the ribosomal polypeptide exit tunnel. Arx1 probes the entire set of inner-ring proteins surrounding the tunnel exit, and the C terminus of Rei1 is deeply inserted into the ribosomal tunnel, where it forms specific contacts along almost its entire length. We provide genetic and biochemical evidence that failure to insert the C terminus of Rei1 precludes subsequent steps of 60S maturation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The initiation of eukaryotic chromosomal DNA replication requires the formation of an active replicative helicase at the replication origins of chromosomes. Yeast Sld3 and its metazoan counterpart treslin are the hub proteins mediating protein associations critical for formation of the helicase. The Sld7 protein interacts with Sld3, and the complex formed is thought to regulate the function of Sld3. Although Sld7 is a non-essential DNA replication protein that is found in only a limited range of yeasts, its depletion slowed the growth of cells and caused a delay in the S phase. Recently, the Mdm2-binding protein was found to bind to treslin in humans, and its depletion causes defects in cells similar to the depletion of Sld7 in yeast, suggesting their functional relatedness and importance during the initiation step of DNA replication. Here, the crystal structure of Sld7 in complex with Sld3 is presented. Sld7 comprises two structural domains. The N-terminal domain of Sld7 binds to Sld3, and the C-terminal domains connect two Sld7 molecules in an antiparallel manner. The quaternary structure of the Sld3-Sld7 complex shown from the crystal structures appears to be suitable to activate two helicase molecules loaded onto replication origins in a head-to-head manner.

Asymmetric ABC (ATP-binding cassette) transporters make up a significant proportion of this important superfamily of integral membrane proteins. These proteins contain one canonical (catalytic) ATP-binding site and a second atypical site with little enzymatic capability. The baker's yeast (Saccharomyces cerevisiae) Pdr5 multidrug transporter is the founding member of the Pdr subfamily of asymmetric ABC transporters, which exist only in fungi and slime moulds. Because these organisms are of considerable medical and agricultural significance, Pdr5 has been studied extensively, as has its medically important homologue Cdr1 from Candida albicans. Genetic and biochemical analyses of Pdr5 have contributed important observations that are likely to be applicable to mammalian asymmetric ABC multidrug transporter proteins, including the basis of transporter promiscuity, the function of the non-catalytic deviant ATP-binding site, the most complete description of an in vivo transmission interface, and the recent discovery that Pdr5 is a molecular diode (one-way gate). In the present review, we discuss the observations made with Pdr5 and compare them with findings from clinically important asymmetric ABC transporters, such as CFTR (cystic fibrosis transmembrane conductance regulator), Cdr1 and Tap1/Tap2.

Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2-adrenergic receptor (β2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process., (Copyright © 2014 John Wiley & Sons, Ltd.)

Eukaryotic translation initiation factor 4G (eIF4G) plays a crucial role in translation initiation, serving as a scaffolding protein binding several other initiation factors, other proteins, and RNA. Binding of eIF4G to the ATP-dependent RNA helicase eukaryotic translation initiation factor 4A (eIF4A) enhances the activity of eIF4A in solution and in crowded environments. Previously, this activity enhancement was solely attributed to eIF4G, conferring a closed, active conformation upon eIF4A. Here we show that eIF4G contains a low-affinity binding site at the entrance to the ATP-binding cleft on eIF4A, suggesting that regulation of the local ATP concentration may be an additional reason for the enhancement in activity.

Sup35p of Saccharomyces cerevisiae can form the [PSI+] prion, an infectious amyloid in which the protein is largely inactive. The part of Sup35p that forms the amyloid is the region normally involved in control of mRNA turnover. The formation of [PSI+] by Sup35p's from other yeasts has been interpreted to imply that the prion-forming ability of Sup35p is conserved in evolution, and thus of survival/fitness/evolutionary value to these organisms. We surveyed a larger number of yeast and fungal species by the same criteria as used previously and find that the Sup35p from many species cannot form prions. [PSI+] could be formed by the Sup35p from Candida albicans, Candida maltosa, Debaromyces hansenii, and Kluyveromyces lactis, but orders of magnitude less often than the S. cerevisiae Sup35p converts to the prion form. The Sup35s from Schizosaccharomyces pombe and Ashbya gossypii clearly do not form [PSI+]. We were also unable to detect [PSI+] formation by the Sup35ps from Aspergillus nidulans, Aspergillus fumigatus, Magnaporthe grisea, Ustilago maydis, or Cryptococcus neoformans. Each of two C. albicans SUP35 alleles can form [PSI+], but transmission from one to the other is partially blocked. These results suggest that the prion-forming ability of Sup35p is not a conserved trait, but is an occasional deleterious side effect of a protein domain conserved for another function., (Copyright © 2014 by the Genetics Society of America.)

Calcium-mediated signaling pathways are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes. In Saccharomyces cerevisiae, at least two different calcium uptake systems have been identified: the high-affinity calcium influx system (HACS) and the low-affinity calcium influx system (LACS). Compared to the HACS, the LACS in fungi is not well known. In this study, FigA, a homolog of the LACS member Fig1 from S. cerevisiae, was functionally characterized in the filamentous fungus Aspergillus nidulans. Loss of figA resulted in retardant hyphal growth and a sharp reduction of conidial production. Most importantly, FigA is essential for the homothallic mating (self-fertilization) process; further, FigA is required for heterothallic mating (outcrossing) in the absence of HACS midA. Interestingly, in a figA deletion mutant, adding extracellular Ca(2+) rescued the hyphal growth defects but could not restore asexual and sexual reproduction. Furthermore, quantitative PCR results revealed that figA deletion sharply decreased the expression of brlA and nsdD, which are known as key regulators during asexual and sexual development, respectively. In addition, green fluorescent protein (GFP) tagging at the C terminus of FigA (FigA::GFP) showed that FigA localized to the center of the septum in mature hyphal cells, to the location between vesicles and metulae, and between the junctions of metulae and phialides in conidiophores. Thus, our findings suggest that FigA, apart from being a member of a calcium uptake system in A. nidulans, may play multiple unexplored roles during hyphal growth and asexual and sexual development.

Protein secretion is an essential process for living organisms. In eukaryotes, this encompasses numerous steps mediated by several hundred cellular proteins. The core functions of translocation through the endoplasmic reticulum membrane, primary glycosylation, folding and quality control, and vesicle-mediated secretion are similar from yeasts to higher eukaryotes. However, recent research has revealed significant functional differences between yeasts and mammalian cells, and even among diverse yeast species. This review provides a current overview of the canonical protein secretion pathway in the model yeast Saccharomyces cerevisiae, highlighting differences to mammalian cells as well as currently unresolved questions, and provides a genomic comparison of the S. cerevisiae pathway to seven other yeast species where secretion has been investigated due to their attraction as protein production platforms, or for their relevance as pathogens. The analysis of Candida albicans, Candida glabrata, Kluyveromyces lactis, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, and Schizosaccharomyces pombe reveals that many - but not all - secretion steps are more redundant in S. cerevisiae due to duplicated genes, while some processes are even absent in this model yeast. Recent research obviates that even where homologous genes are present, small differences in protein sequence and/or differences in the regulation of gene expression may lead to quite different protein secretion phenotypes., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

The methylotrophic yeast Pichia pastoris is an attractive platform for a plethora of recombinant proteins. There is growing evidence that host cells producing recombinant proteins are exposed to a variety of cellular stresses resulting in the induction of the unfolded protein response (UPR) pathway. At present, there is only limited information about the cellular reactions of the host cells at the level of the proteome, especially with regard to recombinant protein secretion. Here we monitored xylanase A secretion from Bacillus halodurans C-125 (xynA) in P. pastoris, using strains containing different copy numbers of the gene encoding xylanase A and co-overexpressing the gene encoding the UPR-regulating transcription factor HAC1 by applying a quantitative proteomics approach (iTRAQ-LC-MS/MS). Many important cellular processes, including carbon metabolism, stress response and protein folding are affected in the investigated conditions. Notably, the analysis revealed that strong over-expression of xynA can efficiently improve protein production but simultaneously cause an unfolded protein burden with a subsequent induction of the UPR. This limits the further improvement of protein production levels. Remarkably, constitutive expression of the gene encoding HAC1 lessens the unfolded protein burden by attenuating protein synthesis and increasing ER protein folding efficiency which is beneficial for protein secretion., Biological Significance: Pichia pastoris expression systems have been successfully used for over 20years in basic research and in the biotechnology industry for the production and secretion of a wide range of recombinant proteins. In particular, secretion of recombinant proteins is still one of the main reasons for using P. pastoris. It has become obvious that many protein products can lead to severe stress on the host cell when being over-expressed, thus limiting the potential yield. Detailed understanding of the physiological responses to such stresses gives rise to engineering of host cells that can better cope with the stress factors. Therefore, the regulatory mechanism of heterologous protein secretion by quantitative mass-spectrometry (MS) proteomics is a growing field and an important endeavor in improving protein annotation. Many important cellular processes, including carbon and amino acid metabolism, stress response and protein folding are affected in the over-expression strains. This data represent a first step towards a systems wide approach to assess the response with recombinant protein induced stress in P. pastoris., (© 2013.)

How do cells direct the microtubule motor protein dynein to move cellular components to the right place at the right time? Recent studies in budding yeast shed light on a new mechanism for directing dynein, involving the protein She1. She1 restricts where and when dynein moves the nucleus and mitotic spindle. Experiments with purified proteins show that She1 binds to microtubules and inhibits dynein by stalling the motor on its track. Here I describe what we have learned so far about She1, based on a combination of genetic, cell biology, and biophysical approaches. These findings set the stage for further interrogation of the She1 mechanism, and raise the question of whether similar mechanisms exist in other species., (© 2013 WILEY Periodicals, Inc.)

Zymolyase (lyticase) is used for cell wall digestion in yeast experiments and is needed for incubation processes under moderate experimental conditions. This has been thought to cause unfavorable effects, and many researchers are aware that the enzyme method is unsuitable for RNA preparation following several reports of stress responses to the enzyme process. However, RNA preparation with enzyme digestion continues to be used. This may be because there have been insufficient data directly comparing RNA preparation conditions with previous studies. We investigated the influence of enzyme processes in RNA preparation using a DNA microarray, and compared superoxide dismutase (SOD) activities with a non-treated control and the results of previous research. Gene expressions were commonly changed by enzyme processes, and SOD activities increased only during short-term incubation. Meanwhile, both SOD gene expressions and SOD activity during RNA preparation indicated different results than gained under conditions of long-term incubation. These results suggest that zymolyase treatment surely influences gene expressions and enzyme activity, although the effect assumed by previous studies is not necessarily in agreement with that of RNA preparation.

The telomere-ending binding protein complex CST (Cdc13-Stn1-Ten1) mediates critical functions in both telomere protection and replication. We devised a co-expression and affinity purification strategy for isolating large quantities of the complete Candida glabrata CST complex. The complex was found to exhibit a 2∶4∶2 or 2∶6∶2 stoichiometry as judged by the ratio of the subunits and the native size of the complex. Stn1, but not Ten1 alone, can directly and stably interact with Cdc13. In gel mobility shift assays, both Cdc13 and CST manifested high-affinity and sequence-specific binding to the cognate telomeric repeats. Single molecule FRET-based analysis indicates that Cdc13 and CST can bind and unfold higher order G-tail structures. The protein and the complex can also interact with non-telomeric DNA in the absence of high-affinity target sites. Comparison of the DNA-protein complexes formed by Cdc13 and CST suggests that the latter can occupy a longer DNA target site and that Stn1 and Ten1 may contact DNA directly in the full CST-DNA assembly. Both Stn1 and Ten1 can be cross-linked to photo-reactive telomeric DNA. Mutating residues on the putative DNA-binding surface of Candida albicans Stn1 OB fold domain caused a reduction in its crosslinking efficiency in vitro and engendered long and heterogeneous telomeres in vivo, indicating that the DNA-binding activity of Stn1 is required for telomere protection. Our data provide insights on the assembly and mechanisms of CST, and our robust reconstitution system will facilitate future biochemical analysis of this important complex., Competing Interests: The authors have declared that no competing interests exist.

Ribosome-associated chaperones act in early folding events during protein synthesis. Structural information is available for prokaryotic chaperones (such as trigger factor), but structural understanding of these processes in eukaryotes lags far behind. Here we present structural analyses of the eukaryotic ribosome-associated complex (RAC) from Saccharomyces cerevisiae and Chaetomium thermophilum, consisting of heat-shock protein 70 (Hsp70) Ssz1 and the Hsp40 Zuo1. RAC is an elongated complex that crouches over the ribosomal tunnel exit and seems to be stabilized in a distinct conformation by expansion segment ES27. A unique α-helical domain in Zuo1 mediates ribosome interaction of RAC near the ribosomal proteins L22e and L31e and ribosomal RNA helix H59. The crystal structure of the Ssz1 ATPase domain bound to ATP-Mg²⁺ explains its catalytic inactivity and suggests that Ssz1 may act before the RAC-associated chaperone Ssb. Our study offers insights into the interplay between RAC, the ER membrane-integrated Hsp40-type protein ERj1 and the signal-recognition particle.

PROPPINs are a family of PtdIns3P and PtdIns(3,5)P 2-binding proteins. The crystal structure now unravels the presence of two distinct phosphoinositide-binding sites at the circumference of the seven bladed β-propeller. Mutagenesis analysis of the binding sites shows that both are required for normal membrane association and autophagic activities. We identified a set of evolutionarily conserved basic and polar residues within both binding pockets, which are crucial for phosphoinositide binding. We expect that membrane association of PROPPINs is further stabilized by membrane insertions and interactions with other proteins.

Background: Kar3Vik1 is a heterodimeric kinesin with one catalytic subunit (Kar3) and one noncatalytic subunit (Vik1)., Results: Vik1 experiences conformational changes in regions analogous to the force-producing elements in catalytic kinesins., Conclusion: A molecular mechanism by which Kar3 could trigger Vik1's release from microtubules was revealed., Significance: These findings will serve as the prototype for understanding the motile mechanism of kinesin-14 motors in general. It is widely accepted that movement of kinesin motor proteins is accomplished by coupling ATP binding, hydrolysis, and product release to conformational changes in the microtubule-binding and force-generating elements of their motor domain. Therefore, understanding how the Saccharomyces cerevisiae proteins Cik1 and Vik1 are able to function as direct participants in movement of Kar3Cik1 and Kar3Vik1 kinesin complexes presents an interesting challenge given that their motor homology domain (MHD) cannot bind ATP. Our crystal structures of the Vik1 ortholog from Candida glabrata may provide insight into this mechanism by showing that its neck and neck mimic-like element can adopt several different conformations reminiscent of those observed in catalytic kinesins. We found that when the neck is α-helical and interacting with the MHD core, the C terminus of CgVik1 docks onto the central β-sheet similarly to the ATP-bound form of Ncd. Alternatively, when neck-core interactions are broken, the C terminus is disordered. Mutations designed to impair neck rotation, or some of the neck-MHD interactions, decreased microtubule gliding velocity and steady state ATPase rate of CgKar3Vik1 complexes significantly. These results strongly suggest that neck rotation and neck mimic docking in Vik1 and Cik1 may be a structural mechanism for communication with Kar3.

Ribosomal proteins are synthesized in the cytoplasm, before nuclear import and assembly with ribosomal RNA (rRNA). Little is known about coordination of nucleocytoplasmic transport with ribosome assembly. Here, we identify a transport adaptor, symportin 1 (Syo1), that facilitates synchronized coimport of the two 5S-rRNA binding proteins Rpl5 and Rpl11. In vitro studies revealed that Syo1 concomitantly binds Rpl5-Rpl11 and furthermore recruits the import receptor Kap104. The Syo1-Rpl5-Rpl11 import complex is released from Kap104 by RanGTP and can be directly transferred onto the 5S rRNA. Syo1 can shuttle back to the cytoplasm by interaction with phenylalanine-glycine nucleoporins. X-ray crystallography uncovered how the α-solenoid symportin accommodates the Rpl5 amino terminus, normally bound to 5S rRNA, in an extended groove. Symportin-mediated coimport of Rpl5-Rpl11 could ensure coordinated and stoichiometric incorporation of these proteins into pre-60S ribosomes.

Kar3, a Saccharomyces cerevisiae microtubule minus-end-directed kinesin-14, dimerizes with either Vik1 or Cik1. The C-terminal globular domain of Vik1 exhibits the structure of a kinesin motor domain and binds microtubules independently of Kar3 but lacks a nucleotide binding site. The only known function of Kar3Vik1 is to cross-link parallel microtubules at the spindle poles during mitosis. In contrast, Kar3Cik1 depolymerizes microtubules during mating but cross-links antiparallel microtubules in the spindle overlap zone during mitosis. A recent study showed that Kar3Vik1 binds across adjacent microtubule protofilaments and uses a minus-end-directed powerstroke to drive ATP-dependent motility. The presteady-state experiments presented here extend this study and establish an ATPase model for the powerstroke mechanism. The results incorporated into the model indicate that Kar3Vik1 collides with the microtubule at 2.4 μm(-1) s(-1) through Vik1, promoting microtubule binding by Kar3 followed by ADP release at 14 s(-1). The tight binding of Kar3 to the microtubule destabilizes the Vik1 interaction with the microtubule, positioning Kar3Vik1 for the start of the powerstroke. Rapid ATP binding to Kar3 is associated with rotation of the coiled-coil stalk, and the postpowerstroke ATP hydrolysis at 26 s(-1) is independent of Vik1, providing further evidence that Vik1 rotates with the coiled coil during the powerstroke. Detachment of Kar3Vik1 from the microtubule at 6 s(-1) completes the cycle and allows the motor to return to its initial conformation. The results also reveal key differences in the ATPase cycles of Kar3Vik1 and Kar3Cik1, supporting the fact that these two motors have distinctive biological functions.

The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and the cytoplasm. The yeast NPC is an eightfold symmetric annular structure composed of ~456 polypeptide chains contributed by ~30 distinct proteins termed nucleoporins. Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N-terminal "FG" repeats containing a Gle2p-binding sequence motif and a NPC targeting domain at its C-terminus. We report the crystal structure of the NPC targeting domain of Candida glabrata Nup116, consisting of residues 882-1034 [CgNup116(882-1034)], at 1.94 Å resolution. The X-ray structure of CgNup116(882-1034) is consistent with the molecular envelope determined in solution by small-angle X-ray scattering. Structural similarities of CgNup116(882-1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiae Nup145N, and human Nup98 are discussed., (Copyright © 2012 Wiley Periodicals, Inc.)

In Saccharomyces cerevisiae, four proteins, Shu1, Shu2, Psy3 and Csm2, form a stable SHU-complex both in vivo and in vitro. These proteins are involved in the early stages of the homologous recombination DNA damage repair process. In this paper, the crystal structure of the Psy3-Csm2 sub-complex is presented at 1.8Å resolution and successfully fitted into our small angle X-ray scattering (SAXS) data of the SHU-complex. Taken together with our electrophoretic mobility shift assay (EMSA) results, a model is proposed for the SHU-protein complex coupled with DNA., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Kinesin-14 motors generate microtubule minus-end-directed force used in mitosis and meiosis. These motors are dimeric and operate with a nonprocessive powerstroke mechanism, but the role of the second head in motility has been unclear. In Saccharomyces cerevisiae, the Kinesin-14 Kar3 forms a heterodimer with either Vik1 or Cik1. Vik1 contains a motor homology domain that retains microtubule binding properties but lacks a nucleotide binding site. In this case, both heads are implicated in motility. Here, we show through structural determination of a C-terminal heterodimeric Kar3Vik1, electron microscopy, equilibrium binding, and motility that at the start of the cycle, Kar3Vik1 binds to or occludes two αβ-tubulin subunits on adjacent protofilaments. The cycle begins as Vik1 collides with the microtubule followed by Kar3 microtubule association and ADP release, thereby destabilizing the Vik1-microtubule interaction and positioning the motor for the start of the powerstroke. The results indicate that head-head communication is mediated through the adjoining coiled coil.

Many protein kinases require phosphorylation at their activation loop for induction of catalysis. Mitogen-activated protein kinases (MAPKs) are activated by a unique mode of phosphorylation, on neighboring Tyrosine and Threonine residues. Whereas many kinases obtain their activation via autophosphorylation, MAPKs are usually phosphorylated by specific, dedicated, MAPK kinases (MAP2Ks). Here we show however, that the yeast MAPK Hog1, known to be activated by the MAP2K Pbs2, is activated in pbs2Δ cells via an autophosphorylation activity that is induced by osmotic pressure. We mapped a novel domain at the Hog1 C-terminal region that inhibits this activity. Removal of this domain provides a Hog1 protein that is partially independent of MAP2K, namely, partially rescues osmostress sensitivity of pbs2Δ cells. We further mapped a short domain (7 amino acid residues long) that is critical for induction of autophosphorylation. Its removal abolishes autophosphorylation, but maintains Pbs2-mediated phosphorylation. This 7 amino acids stretch is conserved in the human p38α. Similar to the case of Hog1, it's removal from p38α abolishes p38α's autophosphorylation capability, but maintains, although reduces, its activation by MKK6. This study joins a few recent reports to suggest that, like many protein kinases, MAPKs are also regulated via induced autoactivation.

Pneumocystis carinii (Pc) undergoes morphological transitions between cysts and trophic forms. We have previously described two Pc serine/threonine kinases, termed PcCbk1 and PcSte20, with PcSte20 belonging to a family of kinases involved in yeast mating, while PcCbk1 is a member of a group of protein kinases involved in regulation of cell cycle, shape, and proliferation. As Pc remains genetically intractable, knowledge on specific substrates phosphorylated by these kinases remains limited. Utilizing the phylogenetic relatedness of Pc to Saccharomyces cerevisiae, we interrogated a yeast proteome microarray containing >4000 purified protein based peptides, leading to the identification of 18 potential PcCbk1 and 15 PcSte20 substrates (Z-score > 3.0). A number of these potential protein substrates are involved in bud site selection, polarized growth, and response to mating α factor and pseudohyphal and invasive growth. Full-length open reading frames suggested by the PcCbk1 and PcSte20 protoarrays were amplified and expressed. These five proteins were used as substrates for PcCbk1 or PcSte20, with each being highly phosphorylated by the respective kinase. Finally, to demonstrate the utility of this method to identify novel PcCbk1 and PcSte20 substrates, we analysed DNA sequence data from the partially complete Pc genome database and detected partial sequence information of potential PcCbk1 kinase substrates PcPxl1 and PcInt1. We additionally identified the potential PcSte20 kinase substrate PcBdf2. Full-length Pc substrates were cloned and expressed in yeast, and shown to be phosphorylated by the respective Pc kinases. In conclusion, the yeast protein microarray represents a novel crossover technique for identifying unique potential Pc kinase substrates., (Copyright © 2011 John Wiley & Sons, Ltd.)

The unfolded protein response (UPR) detects the accumulation of unfolded proteins in the endoplasmic reticulum (ER) and adjusts the protein-folding capacity to the needs of the cell. Under conditions of ER stress, the transmembrane protein Ire1 oligomerizes to activate its cytoplasmic kinase and ribonuclease domains. It is unclear what feature of ER stress Ire1 detects. We found that the core ER-lumenal domain (cLD) of yeast Ire1 binds to unfolded proteins in yeast cells and to peptides primarily composed of basic and hydrophobic residues in vitro. Mutation of amino acid side chains exposed in a putative peptide-binding groove of Ire1 cLD impaired peptide binding. Peptide binding caused Ire1 cLD oligomerization in vitro, suggesting that direct binding to unfolded proteins activates the UPR.

Sil1 functions as a NEF (nucleotide-exchange factor) for the ER (endoplasmic reticulum) Hsp70 (heat-shock protein of 70 kDa) Bip in eukaryotic cells. Sil1 may catalyse the ADP release from Bip by interacting directly with the ATPase domain of Bip. In the present study we show the complex crystal structure of the yeast Bip and the NEF Sil1 at the resolution of 2.3 Å (1 Å=0.1 nm). In the Sil1-Bip complex structure, the Sil1 molecule acts as a 'clamp' which binds lobe IIb of the Bip ATPase domain. The binding of Sil1 causes the rotation of lobe IIb ~ 13.5° away from the ADP-binding pocket. The complex formation also induces lobe Ib to swing in the opposite direction by ~ 3.7°. These conformational changes open up the nucleotide-binding pocket in the Bip ATPase domain and disrupt the hydrogen bonds between Bip and bound ADP, which may catalyse ADP release. Mutation of the Sil1 residues involved in binding the Bip ATPase domain compromise the binding affinity of Sil1 to Bip, and these Sil1 mutants also abolish the ability to stimulate the ATPase activity of Bip.

Cyclin-dependent kinases (CDKs) play crucial roles in promoting DNA replication and preventing rereplication in eukaryotic cells [1-4]. In budding yeast, CDKs promote DNA replication by phosphorylating two proteins, Sld2 and Sld3, which generates binding sites for pairs of BRCT repeats (breast cancer gene 1 [BRCA1] C terminal repeats) in the Dpb11 protein [5, 6]. The Sld3-Dpb11-Sld2 complex generated by CDK phosphorylation is required for the assembly and activation of the Cdc45-Mcm2-7-GINS (CMG) replicative helicase. In response to DNA replication stress, the interaction between Sld3 and Dpb11 is blocked by the checkpoint kinase Rad53 [7], which prevents late origin firing [7, 8]. Here we show that the two key CDK sites in Sld3 are conserved in the human Sld3-related protein Treslin/ticrr and are essential for DNA replication. Moreover, phosphorylation of these two sites mediates interaction with the orthologous pair of BRCT repeats in the human Dpb11 ortholog, TopBP1. Finally, we show that DNA replication stress prevents the interaction between Treslin/ticrr and TopBP1 via the Chk1 checkpoint kinase. Our results indicate that Treslin/ticrr is a genuine ortholog of Sld3 and that the Sld3-Dpb11 interaction has remained a critical nexus of S phase regulation through eukaryotic evolution., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The unconventional splicing of Hac1 by the ribonuclease Ire1 is a key event in the activation of the unfolded protein response (UPR) in Saccharomyces cerevisiae. This splicing is independent of the spliceosome and is mediated by a secondary structure at the intron-exon boundaries of the mRNA. Similar unconventional splicing was also described for the gene Xbp1 in human, mouse, C. elegans and D. melanogaster, and for Hac1 in five other fungi. We used reported RNA structures to build a multiple sequence alignment and the Infernal package to search for homologous structures. We identified homologous non-canonical intron structures in 128 out of 156 searched eukaryotic genomes. Our results show that the sequence of the Hac1/Xbp1 intron is highly conserved only around the splice sites recognized by Ire1. The consensus structure of the Hac1/Xbp1 mRNA is well conserved in Fungi and Metazoa and resembles structures previously described. We show that a typical Hac1/Xbp1 intron is very short, only 20-26 bases, whereas yeast species have a long intron (> 100 bases). We identified six species with unambiguous Hac1/Xbp1 homologs that have lost the non-canonical intron structure. We propose that these species use a different mechanism to regulate the UPR.

Prion proteins misfold and aggregate into multiple infectious strain variants that possess unique abilities to overcome prion species barriers, yet the structural basis for the species-specific infectivities of prion strains is poorly understood. Therefore, we have investigated the site-specific structural properties of a promiscuous chimeric form of the yeast prion Sup35 from Saccharomyces cerevisiae and Candida albicans. The Sup35 chimera forms two strain variants, each of which selectively infect one species but not the other. Importantly, the N-terminal and middle domains of the Sup35 chimera (collectively referred to as Sup35NM) contain two prion recognition elements (one from each species) that regulate the nucleation of each strain. Mutations in either prion recognition element significantly bias nucleation of one strain conformation relative to the other. Herein, we have investigated the folding of each prion recognition element for the serine-to-arginine mutant at residue 17 of Sup35NM chimera known to promote nucleation of C. albicans strain conformation. Using cysteine-specific labeling analysis, we find that residues in the C. albicans prion recognition element are solvent-shielded, while those outside the recognition sequence (including most of those in the S. cerevisiae recognition element) are solvent-exposed. Moreover, we find that proline mutations in the C. albicans recognition sequence disrupt the prion templating activity of this strain conformation. Our structural findings reveal that differential folding of complementary and non-complementary prion recognition elements within the prion amyloid core of the Sup35NM chimera is the structural basis for its species-specific templating activity.

Centrosomes organize the bipolar mitotic spindle, and centrosomal defects cause chromosome instability. Protein phosphorylation modulates centrosome function, and we provide a comprehensive map of phosphorylation on intact yeast centrosomes (18 proteins). Mass spectrometry was used to identify 297 phosphorylation sites on centrosomes from different cell cycle stages. We observed different modes of phosphoregulation via specific protein kinases, phosphorylation site clustering, and conserved phosphorylated residues. Mutating all eight cyclin-dependent kinase (Cdk)-directed sites within the core component, Spc42, resulted in lethality and reduced centrosomal assembly. Alternatively, mutation of one conserved Cdk site within γ-tubulin (Tub4-S360D) caused mitotic delay and aberrant anaphase spindle elongation. Our work establishes the extent and complexity of this prominent posttranslational modification in centrosome biology and provides specific examples of phosphorylation control in centrosome function.

A remarkable feature of prion biology is that the same prion protein can misfold into more than one infectious conformation, and these conformations in turn lead to distinct heritable prion strains with different phenotypes. The yeast prion [PSI(+)] is a powerful system for studying how changes in strain conformation affect cross-species transmission. We have previously established that a chimera of the Saccharomyces cerevisiae (SC) and Candida albicans (CA) Sup35 prion domains can cross the SC/CA species barrier in a strain-dependent manner. In vitro, the conversion of the monomeric chimera into the prion (amyloid) form can be seeded by either SC or CA Sup35 amyloid fibers, resulting in two strains: Chim[SC] and Chim[CA]. These strains have a "molecular memory" of their originating species in that Chim[SC] preferentially seeds the conversion of SC Sup35, and vice versa. To investigate how this species specificity is conformationally encoded, we used amide exchange and limited proteolysis to probe the structures of these two strains. We found that the amyloid cores of Chim[SC] and Chim[CA] are predominantly confined to the SC-derived and CA-derived residues, respectively. In addition, the chimera is able to propagate the Chim[CA] conformation even when the SC residues comprising the Chim[SC] core were deleted. Thus, the two strains have non-overlapping and modular amyloid cores that determine whether SC or CA residues are presented on the growing face of the prion seed. These observations establish how conformations determine the specificity of prion transmission and demonstrate a remarkable plasticity to amyloid misfolding., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Pub1p, a highly abundant poly(A)+ mRNA binding protein in Saccharomyces cerevisiae, influences the stability and translational control of many cellular transcripts, particularly under some types of environmental stresses. We have studied the structure, RNA and protein recognition modes of different Pub1p constructs by NMR spectroscopy. The structure of the C-terminal RRM domain (RRM3) shows a non-canonical N-terminal helix that packs against the canonical RRM fold in an original fashion. This structural trait is conserved in Pub1p metazoan homologues, the TIA-1 family, defining a new class of RRM-type domains that we propose to name TRRM (TIA-1 C-terminal domain-like RRM). Pub1p TRRM and the N-terminal RRM1-RRM2 tandem bind RNA with high selectivity for U-rich sequences, with TRRM showing additional preference for UA-rich ones. RNA-mediated chemical shift changes map to β-sheet and protein loops in the three RRMs. Additionally, NMR titration and biochemical in vitro cross-linking experiments determined that Pub1p TRRM interacts specifically with the N-terminal region (1-402) of yeast eIF4G1 (Tif4631p), very likely through the conserved Box1, a short sequence motif neighbouring the Pab1p binding site in Tif4631p. The interaction involves conserved residues of Pub1p TRRM, which define a protein interface that mirrors the Pab1p-Tif4631p binding mode. Neither protein nor RNA recognition involves the novel N-terminal helix, whose functional role remains unclear. By integrating these new results with the current knowledge about Pub1p, we proposed different mechanisms of Pub1p recruitment to the mRNPs and Pub1p-mediated mRNA stabilization in which the Pub1p/Tif4631p interaction would play an important role.

β-barrel membrane proteins in the mitochondrial outer membrane use the TOM40 complex to enter mitochondria and then the TOB/SAM complex to be assembled into the outer membrane. Tom7, a subunit of the TOM40 complex, regulates association of Mdm10 with the TOB complex. Here, we analyzed the role of Tom7 in assembly of β-barrel proteins, including Tom40, a central channel subunit of the TOM40 complex, and porin. Depletion of Tom7 decreased transient accumulation of Tom40 at the level of the TOB complex and retarded assembly of porin in vitro. On the other hand, overexpression of Tom7 resulted in enhanced accumulation of in vitro imported Tom40 in the TOB complex, yet it did not affect the in vitro assembly of porin. Site-specific photocross-linking in vivo revealed that Tom7 directly interacts with Tom40 through its transmembrane segment and with Mdm10. These results collectively show that Tom7 recruits Mdm10, enhancing its association with the MMM1 complex, to regulate timing of the release of Tom40 from the TOB complex for subsequent assembly into the TOM40 complex.

Seipin is a transmembrane protein that resides in the endoplasmic reticulum and concentrates at junctions between the ER and cytosolic lipid droplets. Mutations in the human seipin gene, including the missense mutation A212P, lead to congenital generalized lipodystrophy (CGL), characterized by the lack of normal adipose tissue and accumulation of fat in liver and muscles. In both yeast and CGL patient fibroblasts, seipin is required for normal lipid droplet morphology; in its absence droplets appear to bud abnormally from the ER. Here we report the first purification and physical characterization of seipin. Yeast seipin is in a large discrete protein complex. Affinity purification demonstrated that seipin is the main if not exclusive protein in the complex. Detergent sucrose gradients in H(2)O, and D(2)O and gel filtration were used to determine the size of the seipin complex and account for detergent binding. Both seipin-myc13 (seipin fused to 13 tandem copies of the myc epitope) expressed from the endogenous promoter and overexpressed seipin-mCherry form ∼500 kDa proteins consisting of about 9 copies of seipin. The yeast orthologue of the human A212P allele forms only smaller complexes and is unstable; we hypothesize that this accounts for its null phenotype in humans. Seipin appears as a toroid by negative staining electron microscopy. We speculate that seipin plays at least a structural role in organizing droplets or in communication between droplets and ER.

The acquisition of the correct folding of membrane proteins is a crucial process that involves several steps from the recognition of nascent protein, its targeting to the endoplasmic reticulum membrane, its insertion, and its sorting to its final destination. Yarrowia lipolytica is a hemiascomycetous dimorphic yeast and an alternative eukaryotic yeast model with an efficient secretion pathway. To better understand the quality control of membrane proteins, we constructed a model system based on the uracil permease. Mutated forms of the permease were stabilized and retained in the cell and made the strains resistant to the 5-fluorouracil drug. To identify proteins involved in the quality control, we separated proteins extracted in nondenaturing conditions on blue native gels to keep proteins associated in complexes. Some gel fragments where the model protein was immunodetected were subjected to mass spectrometry analysis. The proteins identified gave a picture of the folding proteome, from the translocation across the endoplasmic reticulum membrane, the folding of the proteins, to the vesicle transport to Golgi or the degradation via the proteasome. For example, EMC complex, Gsf2p or Yet3p, chaperone membrane proteins of the endoplasmic reticulum were identified in the Y. lipolytica native proteome.

The transport activity and substrate specificity of two chimeras consisting of S. cerevisiae Nha1p's N-terminal regions (either first 125 or 184 AA) and the rest of the C. glabrata Cnh1p (up to the total protein length of 946 AA) were compared with those of the two native antiporters. Both chimeric transporters were functional upon expression in S. cerevisiae cells, their presence improved the ability of cells to grow in the presence of high external concentration of K(+), Na(+) or Rb(+) (as chlorides), but not in the presence of the smallest cation (Li(+)). Cation efflux confirmed the ability of chimeras to export cations and showed their significantly reduced transport capacity compared to the wild-type proteins. Despite the very high level of primary sequence identity (87 %) between the S. cerevisiae and C. glabrata plasma-membrane Na(+)/H(+) antiporters, various parts of these proteins are not exchangeable without affecting the antiporter's transport capacity.

Proteins containing ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains interact with various binding partners and function as hubs during ubiquitin-mediated protein degradation. A common interaction of the budding yeast UBL-UBA proteins Rad23 and Dsk2 with the E4 ubiquitin ligase Ufd2 has been described in endoplasmic reticulum-associated degradation among other pathways. The UBL domains of Rad23 and Dsk2 play a prominent role in this process by interacting with Ufd2 and different subunits of the 26 S proteasome. Here, we report crystal structures of Ufd2 in complex with the UBL domains of Rad23 and Dsk2. The N-terminal UBL-interacting region of Ufd2 exhibits a unique sequence pattern, which is distinct from any known ubiquitin- or UBL-binding domain identified so far. Residue-specific differences exist in the interactions of these UBL domains with Ufd2, which are coupled to subtle differences in their binding affinities. The molecular details of their differential interactions point to a role for adaptive evolution in shaping these interfaces.

Grp170 and Hsp110 proteins constitute two evolutionary distinct branches of the Hsp70 family that share the ability to function as nucleotide exchange factors (NEFs) for canonical Hsp70s. Although the NEF mechanism of the cytoplasmic Hsp110s is well understood, little is known regarding the mechanism used by Grp170s in the endoplasmic reticulum. In this study, we compare the yeast Grp170 Lhs1 with the yeast Hsp110 Sse1. We find that residues important for Sse1 NEF activity are conserved in Lhs1 and that mutations in these residues in Lhs1 compromise NEF activity. As previously reported for Sse1, Lhs1 requires ATP to trigger nucleotide exchange in its cognate Hsp70 partner Kar2. Using site-specific cross-linking, we show that the nucleotide-binding domain (NBD) of Lhs1 interacts with the NBD of Kar2 face to face, and that Lhs1 contacts the side of the Kar2 NBD via its protruding C-terminal alpha-helical domain. To directly address the mechanism of nucleotide exchange, we have compared the hydrogen-exchange characteristics of a yeast Hsp70 NBD (Ssa1) in complex with either Sse1 or Lhs1. We find that Lhs1 and Sse1 induce very similar changes in the conformational dynamics in the Hsp70. Thus, our findings demonstrate that despite some differences between Hsp110 and Grp170 proteins, they use a similar mechanism to trigger nucleotide exchange.

Type 2C protein phosphatases (PP2C) are monomeric enzymes and their activities require the presence of magnesium or manganese ions. There are seven PP2C genes, ScPTC1, ScPTC2, ScPTC3, ScPTC4, ScPTC5, ScPTC6 and ScPTC7, in Saccharomyces cerevisiae. PTC6 is highly conserved in pathogenic and nonpathogenic yeasts. In the current study we have demonstrated that the Candida albicans CaPTC6 gene could complement the functions of ScPTC6 in the rapamycin and caffeine sensitivities of S. cerevisiae cells, indicating that they are functional homologues. We have also demonstrated that the CaPTC6-encoded protein is a typical PP2C enzyme and that CaPtc6p is localized in the mitochondrion of yeast-form and hyphal cells. However, deletion of CaPTC6 neither affects cell and hyphal growth nor renders Candida cells sensitive to rapamycin and caffeine. Therefore, possibly with a functional redundancy to other mitochondrial phosphatases, CaPtc6p is likely to be involved in the regulation of a mitochondrial physiology., (Copyright 2009 John Wiley & Sons, Ltd.)

Get3, Get4, and Get5 in Saccharomyces cerevisiae participate in the insertion of tail-anchored proteins into the endoplasmic reticulum membrane. We elucidated the interaction between Get4 and Get5 and investigated their interaction with Get3 and a tetratricopeptide repeat-containing protein, Sgt2. Based on co-immunoprecipitation and crystallographic studies, Get4 and Get5 formed a tight complex, suggesting that they constitute subunits of a larger complex. In contrast, although Get3 interacted physically with the Get4-Get5 complex, low amounts of Get3 co-precipitated with Get5, implying a transient interaction between Get3 and Get4-Get5. Sgt2 also interacted with Get5, although the amount of Sgt2 that co-precipitated with Get5 varied. Moreover, GET3, GET4, and GET5 interacted genetically with molecular chaperone YDJ1, suggesting that chaperones might also be involved in the insertion of tail-anchored proteins.