Fal1p is an essential DEAD-box protein involved in 40S-ribosomal-subunit biogenesis in Saccharomyces cerevisiae

A previously uncharacterized Saccharomyces cerevisiae gene, FAL1, was found by sequence comparison as a homolog of the eukaryotic translation initiation factor 4A (eIF4A). Fal1p has 55% identity and 73% similarity on the amino acid level to yeast eIF4A, the prototype of ATP-dependent RNA helicases of the DEAD-box protein family. Although clearly grouped in the eIF4A subfamily, the essential Fal1p displays a different subcellular function and localization. An HA epitope-tagged Fal1p is localized predominantly in the nucleolus. Polysome analyses in a temperature-sensitive fal1-1 mutant and a Fal1p-depleted strain reveal a decrease in the number of 40S ribosomal subunits. Furthermore, these strains are hypersensitive to the aminoglycoside antibiotics paromomycin and neomycin. Pulse-chase labeling of pre-rRNA and steady-state-level analysis of pre-rRNAs and mature rRNAs by Northern hybridization and primer extension in the Fal1p-depleted strain show that Fal1p is required for pre-rRNA processing at sites A0, A1, and A2. Consequently, depletion of Fal1p leads to decreased 18S rRNA levels and to an overall deficit in 40S ribosomal subunits. Together, these results implicate Fal1p in the 18S rRNA maturation pathway rather than in translation initiation.     

Saccharomyces cerevisiae Nip7p is required for efficient 60S ribosome subunit biogenesis

The Saccharomyces cerevisiae temperature-sensitive (ts) allele nip7-1 exhibits phenotypes associated with defects in the translation apparatus, including hypersensitivity to paromomycin and accumulation of halfmer polysomes. The cloned NIP7+ gene complemented the nip7-1 ts growth defect, the paromomycin hypersensitivity, and the halfmer defect. NIP7 encodes a 181-amino-acid protein (21 kDa) with homology to predicted products of open reading frames from humans, Caenorhabditis elegans, and Arabidopsis thaliana, indicating that Nip7p function is evolutionarily conserved. Gene disruption analysis demonstrated that NIP7 is essential for growth. A fraction of Nip7p cosedimented through sucrose gradients with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Nip7p was found evenly distributed throughout the cytoplasm and nucleus by indirect immunofluorescence; however, in vivo localization of a Nip7p-green fluorescent protein fusion protein revealed that a significant amount of Nip7p is present inside the nucleus, most probably in the nucleolus. Depletion of Nip7-1p resulted in a decrease in protein synthesis rates, accumulation of halfmers, reduced levels of 60S subunits, and, ultimately, cessation of growth. Nip7-1p-depleted cells showed defective pre-rRNA processing, including accumulation of the 35S rRNA precursor, presence of a 23S aberrant precursor, decreased 20S pre-rRNA levels, and accumulation of 27S pre-rRNA. Delayed processing of 27S pre-rRNA appeared to be the cause of reduced synthesis of 25S rRNA relative to 18S rRNA, which may be responsible for the deficit of 60S subunits in these cells.     

Identification of a new nuclear gene (CEM1) encoding a protein homologous to a beta-keto-acyl synthase which is essential for mitochondrial respiration in Saccharomyces cerevisiae

We have analysed a new gene, CEM1, from Saccharomyces cerevisiae. Inactivation of this gene leads to a respiratory-deficient phenotype. The deduced protein sequence shows strong similarities with beta-keto-acyl synthases or condensing enzymes. Typically, enzymes of this class are involved in the synthesis of fatty acids or similar molecules. An analysis of the mitochondrial lipids and fatty acids shows no major difference between the wild type and deleted strains, implying that the CEM1 gene product is not involved in the synthesis of the bulk fatty acids. Thus it is possible that the CEM1 protein is involved in the synthesis of a specialized molecule, probably related to a fatty acid, which is essential for mitochondrial respiration.     

Mlc1p is a light chain for the unconventional myosin Myo2p in Saccharomyces cerevisiae

For decades, the fluid-filled lung has been a valuable research model for understanding normal and abnormal pulmonary physiology. It has lagged behind, however, as a useful therapeutic tool. Recently, the potential applications of perflubron's physicochemical and biologic properties have been realized. In animal models of several types of hypoxic respiratory failure, perflubron's efficacy in improving gas exchange and compliance has been demonstrated. Preliminary clinical studies of PLV in neonates who have RDS and CDH, and in children and adults who have ARDS have shown promise. Pivotal prospective, controlled studies have yet to be completed.     

A novel DNA-binding protein bound to the mitochondrial inner membrane restores the null mutation of mitochondrial histone Abf2p in Saccharomyces cerevisiae

The yeast mitochondrial HMG-box protein, Abf2p, is essential for maintenance of the mitochondrial genome. To better understand the role of Abf2p in the maintenance of the mitochondrial chromosome, we have isolated a multicopy suppressor (YHM2) of the temperature-sensitive defect associated with an abf2 null mutation. The function of Yhm2p was characterized at the molecular level. Yhm2p has 314 amino acid residues, and the deduced amino acid sequence is similar to that of a family of mitochondrial carrier proteins. Yhm2p is localized in the mitochondrial inner membrane and is also associated with mitochondrial DNA in vivo. Yhm2p exhibits general DNA-binding activity in vitro. Thus, Yhm2p appears to be novel in that it is a membrane-bound DNA-binding protein. A sequence that is similar to the HMG DNA-binding domain is important for the DNA-binding activity of Yhm2p, and a mutation in this region abolishes the ability of YHM2 to suppress the temperature-sensitive defect of respiration of the abf2 null mutant. Disruption of YHM2 causes a significant growth defect in the presence of nonfermentable carbon sources such as glycerol and ethanol, and the cells have defects in respiration as determined by 2,3,5,-triphenyltetrazolium chloride staining. Yhm2p may function as a member of the protein machinery for the mitochondrial inner membrane attachment site of mitochondrial DNA during replication and segregation of mitochondrial genomes.     

De novo protein synthesis is essential for thermotolerance acquisition in a Saccharomyces cerevisiae trehalose synthase mutant

Heat shock (25 degrees C to 37 degrees C for 30 min) acquisition of thermotolerance (at 50 degrees C) was observed in a yeast trehalose synthase mutant and the corresponding control strain. The acquisition of thermotolerance in the control strain was maintained for a significantly longer time than in the trehalose synthase mutant. The heat shock was associated with the synthesis of specific heat shock proteins and, in the case of the control strain, also trehalose accumulation. Inhibition of protein synthesis during the heat shock totally abolished acquisition of thermotolerance in both strains but not trehalose accumulation in the control. It was concluded that trehalose may only be required for prolonged stress protection while heat shock proteins are required for heat shock acquisition of thermotolerance.     

The PY-motif of Bul1 protein is essential for growth of Saccharomyces cerevisiae under various stress conditions

The effects of chronic maternal administration of ethanol on nitric oxide synthase (NOS) activity and the numbers of NOS containing neurons, and CA1 and CA3 pyramidal neurons in the hippocampus of the near term fetal guinea pig at gestational day (GD) 62 were investigated. Pregnant guinea pigs received oral administration of 4 g ethanol/kg maternal body weight (n = 5), isocaloric sucrose/pair feeding (n = 5) or water (n = 5), or no treatment (NT; n = 5) from GD 2 to GD 61. NOS activity in the 25,000 x g supernatant of hippocampal homogenate was determined using a radiometric assay. The numbers of NOS containing neurons, and CA1 and CA3 pyramidal neurons were determined using NADPH diaphorase histochemistry and cresyl violet staining, respectively. The chronic ethanol regimen produced a maternal blood ethanol concentration of 193 +/- 13 mg/dl at 1 h after the second divided dose on GD 57. Chronic ethanol exposure produced fetal body, brain, and hippocampal growth restriction and decreased fetal hippocampal NOS activity compared with the isocaloric sucrose/pair feeding, water, and NT experimental groups, but did not affect the number of NOS containing and CA1 or CA3 pyramidal neurons. These data demonstrate that, in the near term fetus, chronic maternal administration of ethanol suppresses hippocampal NOS activity and consequent formation of NO, without loss of NOS containing neurons and prior to loss of CA1 pyramidal neurons that occurs in the adult.     

The ARG11 gene of Saccharomyces cerevisiae encodes a mitochondrial integral membrane protein required for arginine biosynthesis

Prototype strain MG409 (arg11-1) is a severe arginine bradytroph with greatly reduced ornithine and arginine pools, although all known enzymes required for arginine biosynthesis are functional. To identify the function required for normal arginine production impaired in MG409, we have cloned, sequenced, and performed a first molecular characterization of ARG11. We show that the ARG11 open reading frame encodes a putative 292-residue protein with a predicted molecular mass of 31.5 kDa. Sequence similarities, a tripartite organization, and six potential hydrophobic transmembrane spans suggest that Arg11p belongs to the mitochondrial integral inner membrane carrier family. We have used immuno-Western blotting and hemagglutinin epitope-tagged derivatives of Arg11p, Arg8p (a mitochondrial matrix marker), and Arg3p (a cytosolic marker) to demonstrate that Arg11p is confined to the mitochondria and behaves like an integral membrane protein. A deletion created in ARG11 causes the same arginine-leaky behavior as the original arg11-1 mutation, which yields a premature stop codon at residue 266. Arg11p thus appears to fulfill a partially redundant function requiring its 27 carboxyl-terminal amino acids. As a working hypothesis, we propose that Arg11p participates in the export of matrix-made ornithine into the cytosol.     

Pet127p, a membrane-associated protein involved in stability and processing of Saccharomyces cerevisiae mitochondrial RNAs

Nuclear mutations that inactivate the Saccharomyces cerevisiae gene PET127 dramatically increased the levels of mutant COX3 and COX2 mitochondrial mRNAs that were destabilized by mutations in their 5' untranslated leaders. The stabilizing effect of pet127 delta mutations occurred both in the presence and in the absence of translation. In addition, pet127 delta mutations had pleiotropic effects on the stability and 5' end processing of some wild-type mRNAs and the 15S rRNA but produced only a leaky nonrespiratory phenotype at 37 degrees C. Overexpression of PET127 completely blocked respiratory growth and caused cells to lose wild-type mitochondrial DNA, suggesting that too much Pet127p prevents mitochondrial gene expression. Epitope-tagged Pet127p was specifically located in mitochondria and associated with membranes. These findings suggest that Pet127p plays a role in RNA surveillance and/or RNA processing and that these functions may be membrane bound in yeast mitochondria.     

Cse1p is required for export of Srp1p/importin-alpha from the nucleus in Saccharomyces cerevisiae

In metazoan cells, the CAS protein has been shown to function as a recycling factor for the importin-alpha subunit of the classical nuclear localization signal receptor, exporting importin-alpha from the nucleus to allow its participation in multiple rounds of nuclear import. CAS is a member of a family of proteins that bear homology to the larger subunit of the nuclear localization signal receptor, importin-beta, and that are found in all eukaryotes from yeast to humans. Sequence similarity identifies the product of the Saccharomyces cerevisiae CSE1 gene as a potential CAS homologue. Here we present evidence that Cse1p is the functional homologue of CAS: Cse1p is required to prevent accumulation of Srp1p/importin-alpha in the nucleus, it localizes to the nuclear envelope in a pattern typical of nuclear transport receptors, and it associates in vivo with Srp1p in a nucleotide-specific manner. We show further that mutations in CSE1 and SRP1 have specific effects on their association and on the intracellular localization of Cse1p.     

PAS10 is a tetratricopeptide-repeat protein that is essential for the import of most matrix proteins into peroxisomes of Saccharomyces cerevisiae

pas mutants of Saccharomyces cerevisiae are disturbed in peroxisome assembly (pas) and proliferation. Here we report the characterization of the PAS10 gene and its product (PAS10) that is essential for the import of a large subset of proteins into the peroxisomal matrix. PAS10, a protein of 69 kDa, is a member of the tetratricopeptide repeat, or snap helix, protein family, characterized by several direct repeats of a degenerate 34-amino acid motif (Sikorski, R. S., Boguski, M. S., Goebl, M. & Hieter, P. (1990) Cell 60, 307-317). Other members of this family are MAS70 (S. cerevisiae) and MOM72 (Neurospora crassa), which are mitochondrial receptors for protein import. A pas10 null mutant accumulates peroxisomal, leaflet-like membrane structures and exhibits deficient import of a number of peroxisomal matrix enzymes, particularly of proteins with an SKL-like import signal. In contrast, 3-ketoacyl-CoA thiolase associated with these membranes is resistant in vitro to degradation by proteinase K, indicating true protein import. These results suggest that PAS10 is an essential component of a peroxisomal import machinery which mediates the translocation of a specific subset of proteins to the peroxisomal matrix with an SKL-like import signal.     

The Saccharomyces cerevisiae prenylcysteine carboxyl methyltransferase Ste14p is in the endoplasmic reticulum membrane

Eukaryotic proteins containing a C-terminal CAAX motif undergo a series of posttranslational CAAX-processing events that include isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. We demonstrated previously that the STE14 gene product of Saccharomyces cerevisiae mediates the carboxyl methylation step of CAAX processing in yeast. In this study, we have investigated the subcellular localization of Ste14p, a predicted membrane-spanning protein, using a polyclonal antibody generated against the C terminus of Ste14p and an in vitro methyltransferase assay. We demonstrate by immunofluorescence and subcellular fractionation that Ste14p and its associated activity are localized to the endoplasmic reticulum (ER) membrane of yeast. In addition, other studies from our laboratory have shown that the CAAX proteases are also ER membrane proteins. Together these results indicate that the intracellular site of CAAX protein processing is the ER membrane, presumably on its cytosolic face. Interestingly, the insertion of a hemagglutinin epitope tag at the N terminus, at the C terminus, or at an internal site disrupts the ER localization of Ste14p and results in its mislocalization, apparently to the Golgi. We have also expressed the Ste14p homologue from Schizosaccharomyces pombe, mam4p, in S. cerevisiae and have shown that mam4p complements a Deltaste14 mutant. This finding, plus additional recent examples of cross-species complementation, indicates that the CAAX methyltransferase family consists of functional homologues.     

Structure-function analyses of the Ssc1p, Mdj1p, and Mge1p Saccharomyces cerevisiae mitochondrial proteins in Escherichia coli

The DnaK, DnaJ, and GrpE proteins of Escherichia coli have been universally conserved across the biological kingdoms and work together to constitute a highly efficient molecular chaperone machine. We have examined the extent of functional conservation of Saccharomyces cerevisiae Ssc1p, Mdj1p, and Mge1p by analyzing their ability to substitute for their corresponding E. coli homologs in vivo. We found that the expression of yeast Mge1p, the GrpE homolog, allowed for the deletion of the otherwise essential grpE gene of E. coli, albeit only up to 40 degrees C. The inability of Mge1p to substitute for GrpE at very high temperatures is consistent with our previous finding that it specifically failed to stimulate DnaK's ATPase at such extreme conditions. In contrast to Mge1p, overexpression of Mdj1p, the DnaJ homolog, was lethal in E. coli. This toxicity was specifically relieved by mutations which affected the putative zinc binding region of Mdj1p. Overexpression of a truncated version of Mdj1p, containing the J- and Gly/Phe-rich domains, partially substituted for DnaJ function at high temperature. A chimeric protein, consisting of the J domain of Mdj1p coupled to the rest of DnaJ, acted as a super-DnaJ protein, functioning even more efficiently than wild-type DnaJ. In contrast to the results with Mge1p and Mdj1p, both the expression and function of Ssc1p, the DnaK homolog, were severely compromised in E. coli. We were unable to demonstrate any functional complementation by Ssc1p, even when coexpressed with its Mdj1p cochaperone in E. coli.     

Saccharomyces cerevisiae Gcs1 is an ADP-ribosylation factor GTPase-activating protein

Movement of material between intracellular compartments takes place through the production of transport vesicles derived from donor membranes. Vesicle budding that results from the interaction of cytoplasmic coat proteins (coatomer and clathrin) with intracellular organelles requires a type of GTP-binding protein termed ADP-ribosylation factor (ARF). The GTPase cycle of ARF proteins that allows the uncoating and fusion of a transport vesicle with a target membrane is mediated by ARF-dependent GTPase-activating proteins (GAPs). A previously identified yeast protein, Gcs1, exhibits structural similarity to a mammalian protein with ARF-GAP activity in vitro. We show herein that the Gcs1 protein also has ARF-GAP activity in vitro using two yeast Arf proteins as substrates. Furthermore, Gcs1 function is needed for the efficient secretion of invertase, as expected for a component of vesicle transport. The in vivo role of Gcs1 as an ARF GAP is substantiated by genetic interactions between mutations in the ARF1/ARF2 redundant pair of yeast ARF genes and a gcs1-null mutation; cells lacking both Gcs1 and Arf1 proteins are markedly impaired for growth compared with cells missing either protein. Moreover, cells with decreased levels of Arf1 or Arf2 protein, and thus with decreased levels of GTP-Arf, are markedly inhibited for growth by increased GCS1 gene dosage, presumably because increased levels of Gcs1 GAP activity further decrease GTP-Arf levels. Thus by both in vitro and in vivo criteria, Gcs1 is a yeast ARF GAP.     

Dbp6p is an essential putative ATP-dependent RNA helicase required for 60S-ribosomal-subunit assembly in Saccharomyces cerevisiae

A previously uncharacterized Saccharomyces cerevisiae open reading frame, YNR038W, was analyzed in the context of the European Functional Analysis Network. YNR038W encodes a putative ATP-dependent RNA helicase of the DEAD-box protein family and was therefore named DBP6 (DEAD-box protein 6). Dbp6p is essential for cell viability. In vivo depletion of Dbp6p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase labeling of pre-rRNA and steady-state analysis of pre-rRNA and mature rRNA by Northern hybridization and primer extension show that Dbp6p depletion leads to decreased production of the 27S and 7S precursors, resulting in a depletion of the mature 25S and 5.8S rRNAs. Furthermore, hemagglutinin epitope-tagged Dbp6p is detected exclusively within the nucleolus. We propose that Dbp6p is required for the proper assembly of preribosomal particles during the biogenesis of 60S ribosomal subunits, probably by acting as an rRNA helicase.     

Mrs5p, an essential protein of the mitochondrial intermembrane space, affects protein import into yeast mitochondria

We have isolated a yeast nuclear gene that suppresses the previously described respiration-deficient mrs2-1 mutation when present on a multicopy plasmid. Elevated gene dosage of this new gene, termed MRS5, suppresses also the pet phenotype of a mitochondrial splicing-deficient group II intron mutation M1301. The MRS5 gene product, a 13-kDa protein of low abundance, shows no similarity to other known proteins and is associated with the inner mitochondrial membrane, protruding into the intermembrane space. MRS5 codes for an essential protein, as the disruption of this gene is lethal even during growth on fermentable carbon sources. Thus, the Mrs5 protein seems to be involved in mitochondrial key functions aside from oxidative energy conservation, which is dispensable in fermenting yeast cells. Depletion of Mrs5p in yeast cells causes accumulation of unprocessed precursors of the mitochondrial hsp60 protein and defects in all cytochrome complexes. These findings suggest an essential role of Mrs5p in mitochondrial biogenesis.     

Localization and cell surface anchoring of the Saccharomyces cerevisiae flocculation protein Flo1p

The Saccharomyces cerevisiae FLO1 gene encodes a large 1,536-amino-acid serine- and threonine-rich protein involved in flocculation. We have assessed the localization of Flo1p by immunoelectron microscopy, and in this study we show that this protein is located in the external mannoprotein layer of the cell wall, at the plasma membrane level and in the periplasm. The protein was also visualized in the endoplasmic reticulum and in the nuclear envelope, indicating that it was secreted through the secretory pathway. The protein was detected by Western blotting in cell wall extracts as a high-molecular-mass (>200 kDa) polydisperse material obviously as a result of extensive N and probably O glycosylation. Flo1p was extracted from cell walls in large amounts by boiling in sodium dodecyl sulfate, suggesting that it is noncovalently anchored to the cell wall network. The membranous forms of Flo1p were shown to be solubilized by phosphatidylinositol-phospholipase C treatment, suggesting that Flo1p is glycosyl phosphatidylinositol (GPI) anchored to this organelle. The expression of truncated forms with the hydrophobic C-terminal domain deleted led to the secretion of the protein in the culture medium. The hydrophobic C terminus, which is a putative GPI anchoring domain, is therefore necessary for the attachment of Flo1p in the cell wall. Deletion analysis also revealed that the N-terminal domain of Flo1p was essential for cellular aggregation. On the whole, our data indicate that Flo1p is a true cell wall protein which plays a direct role in cell-cell interaction.     

Pex17p of Saccharomyces cerevisiae is a novel peroxin and component of the peroxisomal protein translocation machinery

The function of the human papillomavirus type 11 (HPV 11) E1--E4 spliced protein is not known. E1--E4 protein in HPV-infected tissue is detected in the cytoplasm of differentiated epithelial cells and as immunoreactive bands corresponding to potential monomers, dimers and trimers in immunoblots. The yeast two-hybrid system was employed to test for self association of the HPV 11 E1--E4 protein. To confirm the results of the yeast two-hybrid experiments, coimmunofluorescence studies of a green fluorescent fusion protein (GFP-E1--E4) and a T7 epitope-tagged E1--E4 protein were performed in C33a keratinocytes. E1--E4 protein was shown to self associate in the yeast two-hybrid system, and this result was confirmed by colocalization of GFP-E1--E4 and T7-E1(wedge)E4 proteins in keratinocytes. Analysis of E1--E4 mutants established that the C-terminus was required for self association and that sequences in the N-terminus influenced the intracellular localization of E1--E4 protein. The intracellular expression patterns of GFP-E1--E4 and GFP-E1--E4 mutants were correlated with E1--E4 binding in the yeast two-hybrid system. Those E1--E4 mutants that did not self associate in the yeast two-hybrid system were detected as diffuse cellular fluorescence when expressed as GFP fusions. In contrast, GFP-E1--E4 was detected as a perinuclear aggregate. All E1--E4 mutants capable of associating with E1--E4 in the yeast two-hybrid system were detected as aggregates when expressed as GFP fusion proteins in keratinocytes.