Yeast ERV2p is the first microsomal FAD-linked sulfhydryl oxidase of the Erv1p/Alrp protein family

Saccharomyces cerevisiae Erv2p was identified previously as a distant homologue of Erv1p, an essential mitochondrial protein exhibiting sulfhydryl oxidase activity. Expression of the ERV2 (essential for respiration and vegetative growth 2) gene from a high-copy plasmid cannot substitute for the lack of ERV1, suggesting that the two proteins perform nonredundant functions. Here, we show that the deletion of the ERV2 gene or the depletion of Erv2p by regulated gene expression is not associated with any detectable growth defects. Erv2p is located in the microsomal fraction, distinguishing it from the mitochondrial Erv1p. Despite their distinct subcellular localization, the two proteins exhibit functional similarities. Both form dimers in vivo and in vitro, contain a conserved YPCXXC motif in their carboxyl-terminal part, bind flavin adenine dinucleotide (FAD) as a cofactor, and catalyze the formation of disulfide bonds in protein substrates. The catalytic activity, the ability to form dimers, and the binding of FAD are associated with the carboxyl-terminal domain of the protein. Our findings identify Erv2p as the first microsomal member of the Erv1p/Alrp protein family of FAD-linked sulfhydryl oxidases. We propose that Erv2p functions in the generation of microsomal disulfide bonds acting in parallel with Ero1p, the essential, FAD-dependent oxidase of protein disulfide isomerase.     

The sulfhydryl oxidase Erv1 is a substrate of the Mia40-dependent protein translocation pathway

The thiol oxidase Erv1 and the redox-regulated receptor Mia40/Tim40 are components of a disulfide relay system which mediates import of proteins into the intermembrane space (IMS) of mitochondria. Here we report that Erv1 requires Mia40 for its import into mitochondria. After passage across the translocase of the mitochondrial outer membrane Erv1 interacts via disulfide bonds with Mia40. Erv1 does not contain twin “CX₃C” or twin “CX₉C” motifs which are crucial for import of typical substrates of this pathway and it does not need two “CX₂C” motifs for import into mitochondria. Thus, Erv1 represents an unusual type of substrate of the Mia40-dependent import pathway.     

Erv2p: characterization of the redox behavior of a yeast sulfhydryl oxidase

The FAD prosthetic group of the ERV/ALR family of sulfhydryl oxidases is housed at the mouth of a 4-helix bundle and communicates with a pair of juxtaposed cysteine residues that form the proximal redox active disulfide. Most of these enzymes have one or more additional distal disulfide redox centers that facilitate the transfer of reducing equivalents from the dithiol substrates of these oxidases to the isoalloxazine ring where the reaction with molecular oxygen occurs. The present study examines yeast Erv2p and compares the redox behavior of this ER luminal protein with the augmenter of liver regeneration, a sulfhydryl oxidase of the mitochondrial intermembrane space, and a larger protein containing the ERV/ALR domain, quiescin-sulfhydryl oxidase (QSOX). Dithionite and photochemical reductions of Erv2p show full reduction of the flavin cofactor after the addition of 4 electrons with a midpoint potential of -200 mV at pH 7.5. A charge-transfer complex between a proximal thiolate and the oxidized flavin is not observed in Erv2p consistent with a distribution of reducing equivalents over the flavin and distal disulfide redox centers. Upon coordination with Zn2+, full reduction of Erv2p requires 6 electrons. Zn2+ also strongly inhibits Erv2p when assayed using tris(2-carboxyethyl)phosphine (TCEP) as the reducing substrate of the oxidase. In contrast to QSOX, Erv2p shows a comparatively low turnover with a range of small thiol substrates, with reduced Escherichia coli thioredoxin and with unfolded proteins. Rapid reaction studies confirm that reduction of the flavin center of Erv2p is rate-limiting during turnover with molecular oxygen. This comparison of the redox properties between members of the ERV/ALR family of sulfhydryl oxidases provides insights into their likely roles in oxidative protein folding.     

Hmi1p from Saccharomyces cerevisiae mitochondria is a structure-specific DNA helicase

Hmi1p is a Saccharomyces cerevisiae mitochondrial DNA helicase that is essential for the maintenance of functional mitochondrial DNA. Hmi1p belongs to the superfamily 1 of helicases and is a close homologue of bacterial PcrA and Rep helicases. We have overexpressed and purified recombinant Hmi1p from Escherichia coli and describe here the biochemical characteristics of its DNA helicase activities. Among nucleotide cofactors, the DNA unwinding by Hmi1p was found to occur efficiently only in the presence of ATP and dATP. Hmi1p could unwind only the DNA substrates with a 3'-single-stranded overhang. The length of the 3'-overhang needed for efficient targeting of the helicase to the substrate depended on the substrate structure. For substrates consisting of duplex DNA with a 3'-single-stranded DNA overhang, at least a 19-nt 3'-overhang was needed. In the case of forked substrates with both 3'- and 5'-overhangs, a 9-nt 3'-overhang was sufficient provided that the 5'-overhang was also 9 nt in length. In flap-structured substrates mimicking the chain displacement structures in DNA recombination process, only a 5-nt 3'-single-stranded DNA tail was required for efficient unwinding by Hmi1p. These data indicate that Hmi1p may be targeted to a specific 3'-flap structure, suggesting its possible role in DNA recombination.     

Improved quantitative and qualitative production of single-domain intrabodies mediated by the co-expression of Erv1p sulfhydryl oxidase

Camelidae single domain antibodies (VHHs) have structural and binding features that render them suitable alternatives to conventional IgG antibodies. VHHs are usually easier to produce as recombinant proteins than other antibody fragments. However, for some of the biotechnological applications for which they have been proposed, such as immunochromatography and assisted-crystallography, large amounts of purified antibodies are necessary, whereas some VHH-fusions with common tags such as GFP and SNAP are poorly expressed in the bacterial periplasm. Here we have shown that the co-expression of Erv1p sulfhydryl oxidase resulted in an astonishing yield increase of VHH-SNAP constructs expressed in the bacterial cytoplasm. The resulting recombinant antibodies were also more stable than the antibodies produced using the same plasmid, but in wild-type bacteria. Using this approach, it was possible to obtain tens of milligram of purified fusion antibodies using a basic flask fermentation protocol. Therefore, the described method represents a valid solution to produce inexpensively large amounts of single domain antibodies for in vitro applications and we expect it will be suitable for the production of other antibody fragments.     

Sem1p is a novel subunit of the 26 S proteasome from Saccharomyces cerevisiae

The 26 S proteasome, which catalyzes degradation of polyubiquitinated proteins, is composed of the 20 S proteasome and the 19 S regulatory particle (RP). The RP is composed of the lid and base subcomplexes and regulates the catalytic activity of the 20 S proteasome. In this study, we carried out affinity purification of the lid and base subcomplexes from the tagged strains of Saccharomyces cerevisiae, and we found that the lid contains a small molecular mass protein, Sem1. The Sem1 protein binds with the 26 S proteasome isolated from a mutant with deletion of SEM1 but not with the 26 S proteasome from the wild type. The lid lacking Sem1 is unstable at a high salt concentration. The 19 S RP was immunoprecipitated together with Sem1 by immunoprecipitation using hemagglutinin epitope-tagged Sem1 as bait. Degradation of polyubiquitinated proteins in vivo or in vitro is impaired in the Sem1-deficient 26 S proteasome. In addition, genetic interaction between SEM1 and RPN10 was detected. The human Sem1 homologue hDSS1 was found to be a functional homologue of Sem1 and capable of interacting with the human 26 S proteasome. The results suggest that Sem1, possibly hDSS1, is a novel subunit of the 26 S proteasome and plays a role in ubiquitin-dependent proteolysis.     

The N-terminal cysteine pair of yeast sulfhydryl oxidase Erv1p is essential for in vivo activity and interacts with the primary redox centre.

Yeast Erv1p is a ubiquitous FAD-dependent sulfhydryl oxidase, located in the intermembrane space of mitochondria. The dimeric enzyme is essential for survival of the cell. Besides the redox-active CXXC motif close to the FAD, Erv1p harbours two additional cysteine pairs. Site-directed mutagenesis has identified all three cysteine pairs as essential for normal function. The C-terminal cysteine pair is of structural importance as it contributes to the correct arrangement of the FAD-binding fold. Variations in dimer formation and unique colour changes of mutant proteins argue in favour of an interaction between the N-terminal cysteine pair with the redox centre of the partner monomer.     

An essential function of the mitochondrial sulfhydryl oxidase Erv1p/ALR in the maturation of cytosolic Fe/S proteins

Biogenesis of Fe/S clusters involves a number of essential mitochondrial proteins. Here, we identify the essential Erv1p of Saccharomyces cerevisia mitochondria as a novel component that is specifically required for the maturation of Fe/S proteins in the cytosol, but not in mitochondria. Furthermore, Erv1p was found to be important for cellular iron homeostasis. The homologous mammalian protein ALR (‘augmenter of liver regeneration’), also termed hepatopoietin, can functionally replace defects in Erv1p and thus represents the mammalian orthologue of yeast Erv1p. Previously, a fragment of ALR was reported to exhibit an activity as an extracellular hepatotrophic growth factor. Both Erv1p and full-length ALR are located in the mitochondrial intermembrane space and represent the first components of this compartment with a role in the biogenesis of cytosolic Fe/S proteins. It is likely that Erv1p/ALR operates downstream of the mitochondrial ABC transporter Atm1p/ABC7/Sta1, which also executes a specific task in this essential biochemical process.     

Pet191 is a cytochrome c oxidase assembly factor in Saccharomyces cerevisiae

The twin-Cx(9)C motif protein Pet191 is essential for cytochrome c oxidase maturation. The motif Cys residues are functionally important and appear to be present in disulfide linkages within a large oligomeric complex associated with the mitochondrial inner membrane. The import of Pet191 differs from that of other twin-Cx(9)C motif class of proteins in being independent of the Mia40 pathway.     

Structure-function analysis of the cuprous oxidase activity in Fet3p from Saccharomyces cerevisiae

The Fet3 protein from Saccharomyces cerevisiae is a multicopper oxidase with specificity toward Fe(II) and Cu(I). Fet3p turnover of Fe(II) supports high affinity iron uptake across the yeast plasma membrane, whereas its turnover of Cu(I) contributes to copper resistance in yeast. The structure of Fet3p has been used to identify possible amino acid residues responsible for this protein's reactivity with Cu(I), and structure-function analyses have confirmed this assignment. Fet3p Met(345) is required for the enzyme's reactivity toward Cu(I). Although the Fet3pM345A mutant exhibits wild type spectral and electrochemical behavior, the kinetic constants for Cu(I) turnover and for single-turnover electron transfer from Cu(I) to the enzyme are significantly reduced. The specificity constant with Cu(I) as substrate is reduced by one-fifth, whereas the electron transfer rate from Cu(I) is reduced 50-fold. This mutation has little effect on the reactivity toward Fe(II), indicating that Met(345) contributes specifically to Fet3p reactivity with the cuprous ion. These kinetic defects render the Fet3pM345A unable to support wild type cellular copper resistance, suggesting that there is a finely tuned copper redox balance at the yeast plasma membrane.     

Saccharomyces cerevisiae acyl-CoA oxidase follows a novel, non-PTS1, import pathway into peroxisomes that is dependent on Pex5p

The peroxisomal protein acyl-CoA oxidase (Pox1p) of Saccharomyces cerevisiae lacks either of the two well characterized peroxisomal targeting sequences known as PTS1 and PTS2. Here we demonstrate that peroxisomal import of Pox1p is nevertheless dependent on binding to Pex5p, the PTS1 import receptor. The interaction between Pex5p and Pox1p, however, involves novel contact sites in both proteins. The interaction region in Pex5p is located in a defined area of the amino-terminal part of the protein outside of the tetratricopeptide repeat domain involved in PTS1 recognition; the interaction site in Pox1p is located internally and not at the carboxyl terminus where a PTS1 is normally found. By making use of pex5 mutants that are either specifically disturbed in binding of PTS1 proteins or in binding of Pox1p, we demonstrate the existence of two independent, Pex5p-mediated import pathways into peroxisomes in yeast as follows: a classical PTS1 pathway and a novel, non-PTS1 pathway for Pox1p.     

The kinesin-related protein Kip1p of Saccharomyces cerevisiae is bipolar.

Kip1p is a mitotic spindle-associated kinesin-related protein in Saccharomyces cerevisiae that participates in spindle pole separation. Here, we define the domain arrangement and polypeptide composition of the Kip1p holoenzyme. Electron microscopy of rotary shadowed Kip1p molecules revealed two globular domains 14 nm in diameter connected by a 73-nm long stalk. When the Kip1p domain homologous to the kinesin motor domain was decorated with an unrelated protein, the diameter of the globular domains at both ends of the stalk increased, indicating that Kip1p is bipolar. Soluble Kip1p isolated from S. cerevisiae cells was homomeric, based on the similarity of the sedimentation coefficients of native Kip1p from S. cerevisiae and Kip1p which was purified after expression in insect cells. The holoenzyme molecular weight was estimated using the sedimentation coefficient and Stokes radius, and was most consistent with a tetrameric composition. Kip1p exhibited an ionic strength-dependent transition in its sedimentation coefficient, revealing a potential regulatory mechanism. The position of kinesin motor-related domains at each end of the stalk may allow Kip1p to cross-link either parallel or antiparallel microtubules during mitotic spindle assembly and pole separation.     

Saccharomyces cerevisiae Mob1p is required for cytokinesis and mitotic exit

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G(1) by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK). Here, we demonstrate that, in addition to mitotic exit, S. cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation. The cytokinesis defect was evident in mob1 mutants under conditions in which there was no mitotic-exit defect. Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble. The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings. The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize in mob1 mutants, suggesting that MOB1 functions subsequent to actomyosin ring assembly. We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis. Localization of Mob1p to the bud neck required CDC3, MEN genes CDC5, CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent of MYO1. The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14 mutants. These results suggest that the MEN functions during the mitosis-to-G(1) transition to control cyclin-CDK inactivation and cytokinesis.     

Inp1p is a peroxisomal membrane protein required for peroxisome inheritance in Saccharomyces cerevisiae

Cells have evolved molecular mechanisms for the efficient transmission of organelles during cell division. Little is known about how peroxisomes are inherited. Inp1p is a peripheral membrane protein of peroxisomes of Saccharomyces cerevisiae that affects both the morphology of peroxisomes and their partitioning during cell division. In vivo 4-dimensional video microscopy showed an inability of mother cells to retain a subset of peroxisomes in dividing cells lacking the INP1 gene, whereas cells overexpressing INP1 exhibited immobilized peroxisomes that failed to be partitioned to the bud. Overproduced Inp1p localized to both peroxisomes and the cell cortex, supporting an interaction of Inp1p with specific structures lining the cell periphery. The levels of Inp1p vary with the cell cycle. Inp1p binds Pex25p, Pex30p, and Vps1p, which have been implicated in controlling peroxisome division. Our findings are consistent with Inp1p acting as a factor that retains peroxisomes in cells and controls peroxisome division. Inp1p is the first peroxisomal protein directly implicated in peroxisome inheritance.     

The Saccharomyces cerevisiae protein Mnn10p/Bed1p is a subunit of a Golgi mannosyltransferase complex

In the yeast Saccharomyces cerevisiae many of the N-linked glycans on cell wall and periplasmic proteins are modified by the addition of mannan, a large mannose-containing polysaccharide. Mannan comprises a backbone of approximately 50 alpha-1,6-linked mannoses to which are attached many branches consisting of alpha-1,2-linked and alpha-1,3-linked mannoses. The initiation and subsequent elongation of the mannan backbone is performed by two complexes of proteins in the cis Golgi. In this study we show that the product of the MNN10/BED1 gene is a component of one of these complexes, that which elongates the backbone. Analysis of interactions between the proteins in this complex shows that Mnn10p, and four previously characterized proteins (Anp1p, Mnn9p, Mnn11p, and Hoc1p) are indeed all components of the same large structure. Deletion of either Mnn10p, or its homologue Mnn11p, results in defects in mannan synthesis in vivo, and analysis of the enzymatic activity of the complexes isolated from mutant strains suggests that Mnn10p and Mnn11p are responsible for the majority of the alpha-1, 6-polymerizing activity of the complex.     

Saccharomyces cerevisiae Bor1p is a boron exporter and a key determinant of boron tolerance

Boron is toxic to living organisms when present in excess. Saccharomyces cerevisiae Bor1p is a plasma membrane protein that decreases the intracellular concentration of boron and confers boron tolerance in yeasts. We investigated the detailed characteristics of boron transport by Bor1p and its roles in boron tolerance. Boron transport assays showed that the bor1 deletion mutant (bor1Delta) accumulates higher intracellular concentrations of boron and has a lower rate of boron export. The bor1Delta showed greater susceptibility to high concentrations of boron than the wild-type strain, and the growth rates of both strains were negatively correlated with the intracellular concentrations of boron. With normal to toxic levels of external boron, green fluorescent protein (GFP)-tagged Bor1p localized to the plasma membrane irrespective of the concentration of boron in the medium. Taken together, these results establish Bor1p as a plasma membrane boron exporter and a key determinant of boron tolerance.     

Mks1p is a regulator of nitrogen catabolism upstream of Ure2p in Saccharomyces cerevisiae.

The supply of nitrogen regulates yeast genes affecting nitrogen catabolism, pseudohyphal growth, and meiotic sporulation. Ure2p of Saccharomyces cerevisiae is a negative regulator of nitrogen catabolism that inhibits Gln3p, a positive regulator of DAL5, and other genes of nitrogen assimilation. Dal5p, the allantoate permease, allows ureidosuccinate uptake (Usa(+)) when cells grow on a poor nitrogen source such as proline. We find that overproduction of Mks1p allows uptake of ureidosuccinate on ammonia and lack of Mks1p prevents uptake of ureidosuccinate or Dal5p expression on proline. Overexpression of Mks1p does not affect cellular levels of Ure2p. An mks1 ure2 double mutant can take up ureidosuccinate on either ammonia or proline. Moreover, overexpression of Ure2p suppresses the ability of Mks1p overexpression to allow ureidosuccinate uptake on ammonia. These results suggest that Mks1p is involved in nitrogen control upstream of Ure2p as follows: NH(3) dash, vertical Mks1p dash, vertical Ure2p dash, vertical Gln3p --> DAL5. Either overproduction of Mks1p or deletion of MKS1 interferes with pseudohyphal growth.     

Sds22p is a subunit of a stable isolatable form of protein phosphatase 1 (Glc7p) from Saccharomyces cerevisiae

Protein phosphatase 1 (PP1) is one of the major protein phosphatases in eukaryotic cells. PP1 activity is believed to be controlled by the interaction of PP1 catalytic subunit with various regulatory subunits. The essential gene GLC7 encodes the PP1 catalytic subunit in Saccharomyces cerevisiae. In this study, full-length GLC7(1-312), C-terminal deletion mutants, and C-terminally poly-his tagged mutants were constructed and expressed in a GLC7 knockout strain of S. cerevisiae. Viability studies of the GLC7 knockout strains carrying the plasmids expressing GLC7 C-terminal deletion mutants and their tagged forms showed that the mutants 1-295 and 1-304 were functional, whereas the mutant 1-245 was not. The C-terminally poly-his tagged Glc7p with and without an N-terminal hemagglutinin (HA) tag was partially purified by immobilized Ni(2+) affinity chromatography and further analyzed by gel filtration and ion exchange chromatography. Phosphatase activity assays, SDS-PAGE, and Western blot analyses of the chromatographic fractions suggested that the Glc7p associated with regulatory subunits in vivo. A 40-kDa protein was copurified with tagged Glc7p through several chromatographic procedures. Monoclonal antibody against the HA tag coimmunoprecipitated the tagged Glc7p and the 40-kDa protein. This protein was further purified by a reverse phase HPLC column. Analysis by CNBr digestion, peptide sequencing, and electrospray mass spectrometry showed that this 40-kDa protein is Sds22p, one of the proteins proposed to be a regulatory subunit of Glc7. These results demonstrate that Sds22p forms a complex with Glc7p and that Sds22p:Glc7p is a stable isolatable form of yeast PP1.     

Reactive sulfhydryl groups in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) is inactivated by several thiol- and vicinal dithiol-specific reagents. Titration experiments of the enzyme with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) show the presence of reactive monothiol and vicinal dithiol groups, whose modifications lead to enzyme inactivation. The enzyme is also inactivated by N-(1-pyrenyl)iodoacetamide (PyrIAM), with a binding stoichiometry of approx. 2 mol per mol of enzyme subunit. A high level of pyrene excimer fluorescence is detected on the labeled enzyme, thus implying the reaction of the reagent with two spatially close sulfhydryl groups in the protein. The carboxykinase is not completely inactivated by different vicinal dithiol-specific reagents, thus implying a catalytically non-essential character for these groups. From substrate protection experiments of the enzyme inactivation by DTNB, PyrIAM and vicinal dithiol-specific reagents, it is concluded that the loss of enzyme activity is caused by the modification of both thiol and vicinal dithiol groups in the substrate binding region.     

The putative Saccharomyces cerevisiae hydrolase Ldh1p is localized to lipid droplets

Here, we report the identification of a novel hydrolase in Saccharomyces cerevisiae. Ldh1p (systematic name, Ybr204cp) comprises the typical GXSXG-type lipase motif of members of the α/β-hydrolase family and shares some features with the peroxisomal lipase Lpx1p. Both proteins carry a putative peroxisomal targeting signal type1 (PTS1) and can be aligned with two regions of homology. While Lpx1p is known as a peroxisomal enzyme, subcellular localization studies revealed that Ldh1p is predominantly localized to lipid droplets, the storage compartment of nonpolar lipids. Ldh1p is not required for the function and biogenesis of peroxisomes, and targeting of Ldh1p to lipid droplets occurs independently of the PTS1 receptor Pex5p.