Delayed accumulation of the yeast G1 cyclins Cln1 and Cln2 and the F-box protein Grr1 in response to glucose

The ability to integrate nutrient availability into cell cycle regulation is critical for the viability of organisms. The Saccharomyces cerevisiae ubiquitin ligase SCF(Grr1) regulates the stability of several proteins that participate in cell division or nutrient sensing. Two of its targets, the cyclins Cln1 and Cln2, accumulate in the presence of glucose. When glucose is added to cells growing asynchronously, we show that the accumulation of the cyclins is a very slow response. We report that the F-box protein Grr1 also accumulates at higher levels in the presence of glucose, and that the response to glucose follows a delayed pattern strikingly similar to that described for Cln1 and Cln2. A model for the regulation of F-box proteins predicts that substrate accumulation could stabilize Grr1. While we found that Grr1 is more stable in cells growing with glucose, we show that the delayed responses to glucose occur independently: Grr1 accumulates in the absence of the cyclins, and vice versa. Thus, our results indicate that this model might not apply to the cyclins and Grr1. Glucose is known to strengthen the interaction of Grr1 with Skp1 in the SCF complex. We hypothesize that glucose could promote the accumulation of Grr1 and its assembly into a SCF complex as a feedback regulation that helps compensate for higher cyclins levels.     

Heat shock transiently enhances the synthesis rate of Sis1p,a ribosome-associated DnaJ protein in the oleagenous yeast Apiotrichum curvatum

DnaJ proteins have been localized in different intracellular compartments of eukaryotes. In Apiotrichum curvatum, a fat-storing yeast, we found a DnaJ homolog associated with ribosomes and large cytosolic complexes as well. Using a plant DnaJ probe and a cDNA library constructed from poly(A)⁺ -RNA of A. curvatum grown on oleate we isolated a SIS1 cDNA coding for a 39·5 kDa protein. The putative protein contains neither a zinc finger motif nor a CAAX motif but is characterized by a J-domain at the N-terminal region and a large G-rich region in the middle part of the molecule. Heat shock applied for 1 h resulted in a pronounced but transient increase of the SIS1 mRNA. An antiserum was raised against the bacterially expressed protein. Cell fractions from A. curvatum were further separated by sedimentation centrifugation on sucrose gradients. Analysing the sub-fractions, we detected Sis1p mainly associated with ribosomes, and with particles sedimenting at approximately 200S. Hsp70 was found to be associated with the 200S fraction. The respective cytosolic A. curvatum Hsp70 cDNA was cloned and sequenced. High salt conditions caused the removal of Hsp70 and Sis1p from the 200S complexes. Mild RNase treatment of the 200S fraction afforded monosomes and 200S complexes unaffected by RNase. Heat shock led to a pronounced increase in the rate of de novo synthesis. However, due to the large pools of Sis1p on ribosomes and large cytosolic complexes, the increase in gene activation did not lead to a significant change of the total amount of Sis1p. Accession numbers are: Y12079 for ACHSP70 and Y12080 for ACSIS1. © 1998 John Wiley & Sons, Ltd.     

Characterization of deletion mutations in the carboxy-terminal peptide-binding domain of the Kar2 protein in Saccharomyces cerevisiae

Hsp70 is structurally composed of three domains, an amino-terminal ATPase domain, a proximal 18 kDa peptide-binding domain and a distal 10 kDa carboxy-terminal (C-terminal) domain. To dissect the functional significance of the distal 10 kDa domain, and the boundary region between the proximal and distal C-terminal domains of Kar2p in vivo in Saccharomyces cerevisiae, we constructed a series of plasmids which were truncated or had internal deletion mutations in this region. We found that all these mutations are recessive, and that the distal 10 kDa C-terminal domain, including the HDEL ER-retention sequence, is not essential for cell growth, although the major role of this 10 kDa C-terminal domain is due to the function of the HDEL ER-retention signal. We also found that the Kar2p region (Thr₄₉₂–Thr₅₁₂), corresponding to the β8-sheet in the peptide-binding domain, which constitutes the bottom plate of the binding pocket in E. coli DnaK, is essential for cell viability, and that the following Kar2p region (Glu₅₁₃–Lys₅₄₂), corresponding to α-helices A and B of E. coli DnaK, which was proposed to compose the lid of the binding pocket, is critical but not essential for yeast cell growth. This was further supported by the fact that the latter deletion showed a fully reversible ts phenotype in its growth and only a slight inhibitory effect on the secretion of α-amylase at non-permissive temperature. © 1998 John Wiley & Sons, Ltd.     

URE3] prion propagation is abolished by a mutation of the primary cytosolic Hsp70 of budding yeast

[URE3] and [PSI(+)] are infectious protein forms of the Saccharomyces cerevisiae Ure2p and Sup35p, respectively. We isolated an allele of SSA2, the primary cytosolic Hsp70, in a screen for mutants unable to maintain [URE3]. Designated ssa2-10, the mutation results in a leucine substitution for proline 395, a conserved residue of the peptide-binding domain. This allele also unexpectedly destabilizes [URE3] in newly formed heterozygotes: [URE3] is either absent in heterozygotes formed by crossing wild-type [URE3] cells with ssa2-10 mutants, or present and fully stable. SSA2 deletion mutants are weakly capable of maintaining [URE3]. The ssa2-10 allele is compatible with propagation of [PSI(+)]. However, in combination with a deletion of SSA1, ssa2-10 eliminates the nonsense-suppression phenotype of [PSI(+)] cells.     

Role of the γ component of preprotoxin in expression of the yeast K1 killer phenotype

K₁ killer strains of Saccharomyces cerevisiae secrete a polypeptide toxin to which they are themselves immune. The α and β components of toxin comprise residues 45–147 and 234–316 of the 316-residue K₁ preprotoxin. The intervening 86-residue segment is called γ. A 26-residue signal peptide is removed on entry into the endoplasmic reticulum. The Kex2 protease excises the toxin components from the 290-residue glycosylated protoxin in a late Golgi compartment. Expression of a cDNA copy of the preprotoxin gene confers the complete K₁ killer phenotype on sensitive cells. We now show that expression of immunity requires that α component and the N-terminal 31 residues of γ. An additional C-terminal extension, either eight residues of γ or three of four unrelated peptides, is also required. Expression of preprotoxin terminating at the α C-terminus, or lacking the γ N-terminal half of γ causes profound but reversible growth inhibition. Inhibition is suppressed in cis by the same 31 residues of γ required for immunity to exocellular toxin in trans, but not by the presence of β. Both immunity and growth inhibition are alleviated by insertions in α that inactivate toxin. Inhibition is not suppressed by kex2, chc1 or kre1 mutations, by growth at higher pH or temperature, or by normal K₁ immunity. Inhibition, therefore, probably does not involve processing of the α toxin component at its N-terminus or release from the cell and binding to glucan receptors. Some insertion and substitution mutations in γ severely reduce toxin secretion without affecting immunity. They are presumed to affect protoxin folding in the endoplasmic reticulum and translocation to the Golgi.     

Sdo1p,the yeast orthologue of Shwachman–Bodian–Diamond syndrome protein,binds RNA and interacts with nuclear rRNA‐processing factors

The Shwachman–Bodian–Diamond syndrome protein (SBDS) is a member of a highly conserved protein family of not well understood function, with putative orthologues found in different organisms ranging from Archaea, yeast and plants to vertebrate animals. The yeast orthologue of SBDS, Sdo1p, has been previously identified in association with the 60S ribosomal subunit and is proposed to participate in ribosomal recycling. Here we show that Sdo1p interacts with nucleolar rRNA processing factors and ribosomal proteins, indicating that it might bind the pre‐60S complex and remain associated with it during processing and transport to the cytoplasm. Corroborating the protein interaction data, Sdo1p localizes to the nucleus and cytoplasm and co‐immunoprecipitates precursors of 60S and 40S subunits, as well as the mature rRNAs. Sdo1p binds RNA directly, suggesting that it may associate with the ribosomal subunits also through RNA interaction. Copyright © 2009 John Wiley & Sons, Ltd.     

Sequence of the yeast protein expression plasmid pEG(KT)

The plasmid pEG(KT) is a widely used plasmid for expressing high levels of GST fusion proteins in the yeast Saccharomyces cerevisiae. Unfortunately, a complete sequence file has been lacking, thus complicating efforts to design cloning projects or to modify the plasmid for other uses (e.g. exchanging selection markers, epitope tags or protease cleavage sites to remove the epitope tag). Here, the complete sequence of the pEG(KT) plasmid is reported, thus facilitating its use. Additionally, its use as a vector backbone for high‐level expression of a TAP‐tagged protein is shown. Copyright © 2009 John Wiley & Sons, Ltd.     

The terminal protein of the linear DNA plasmid pGKL2 shares an N-terminal domain of the plasmid-encoded DNA polymerase

The 36K protein attached at the 5′ end of the linear DNA plasmid pGKL2 from the yeast Kluyveromyces lactis was first purified and characterized. The terminal protein was purified from cells (1 kg wet weight) by ammonium sulphate precipitation and two rounds of centrifugation to equilibrium in CsCl gradients. The pGKL2 was present only in the post-microsomal supernatant. Approximately 10 mg of the purified pGKL2 was recovered and digested with DNase I. The terminal protein (final ca. 0·8 mg) was homogeneous by electrophoresis and we determined the N-terminal amino acid sequence up to ten residues, showing that it existed in the cryptic N-terminal domain of pGKL2-ORF2 (DNA polymerase) sequence.     

Physiological effects of unassembled chaperonin Cct subunits in the yeast Saccharomyces cerevisiae

Eukaryotic chaperonins, the Cct complexes, are assembled into two rings, each of which is composed of a stoichiometric array of eight different subunits, which are denoted Cct1p-Cct8p. Overexpression of a single CCT gene in Saccharomyces cerevisiae causes an increase of the corresponding Cct subunit, but not of the Cct complex. Nevertheless, overexpression of certain Cct subunits, especially CCT6, suppresses a wide range of abnormal phenotypes, including those caused by the diverse types of conditional mutations tor2-21, lst8-2 and rsp5-9 and those caused by the concomitant overexpression of Sit4p and Sap155p. The examination of 73 altered forms of Cct6p revealed that the cct6-24 mutation, containing GDGTT --> AAAAA replacements of the conserved ATP-binding motif, was unable to suppress any of these traits, although the cct6-24 allele was completely functional for growth. These results provide evidence for functional differences among Cct subunits and for physiological properties of unassembled subunits. We suggest that the suppression is due to the competition of specific Cct subunits for activities that normally modify various cellular components. Furthermore, we also suggest that the Cct subunits can act as suppressors only in certain states, such as when associated with ATP.     

Mapping of the ACC1/FAS3 gene to the right arm of chromosome XIV of Saccharomyces cerevisiae

The ACC1/FAS3 gene has been mapped to the right arm of chromosome XIV by both genetic and physical methods. The gene is closely linked to RNA2 and is allelic to the ABP2 gene of chromosome XIV.     

Predominant localization of non-specific lipid-transfer protein of the yeast Candida tropicalis in the matrix of peroxisomes

PXP-18 is a 14-kDa major peroxisomal protein of the yeast Candida tropicalis and a homologue of the non-specific lipid-transfer protein (nsLTP) of mammals. Mammalian nsLTP is thought to facilitate the contact of membranes, to stimulate lipid-transfer between them. If PXP-18 functions like nsLTP, it must be present on organelle membranes. Immunoelectron microscopy of C. tropicalis cells indicated that gold particles, which visualized PXP-18, localized exclusively in the matrix of peroxisomes. Subcellular fractionation followed by Western blotting revealed the association of PXP-18 with peroxisomes in C. tropicalis cells. An enzyme-linked immunosorbent assay revealed that almost all the PXP-18 associated with peroxisomes was detectable after the solubilization of the organelle but not before, implying the predominance of PXP-18 inside peroxisomes. This differential assay was applied to the intracellular import of the intact and truncated PXP-18s expressed in Saccharomyces cerevisiae cells. Most of the intact PXP-18 was shown to be imported into the matrix of host-cell peroxisomes, whereas the truncated PXP-18, which lacked the C-terminal tripeptide Pro-Lys-Leu, no longer targeted peroxisomes. These results are consistent with the view that PXP-18 is the matrix protein of peroxisomes and must function in a system other than that of lipid transfer.     

New selectable host–marker systems for multiple genetic manipulations based on TRP1, MET2 and ADE2 in the methylotrophic yeast Hansenula polymorpha

Interest has been increasing in the thermotolerant methylotrophic yeast Hansenula polymorpha as a useful system for fundamental research and applied purposes. Only a few genetic marker genes and auxotrophic hosts are yet available for this yeast. Here we isolated and developed H. polymorpha TRP1, MET2 and ADE2 genes as selectable markers for multiple genetic manipulations. The H. polymorpha TRP1 (HpTRP1), MET2 (HpMET2) and ADE2 (HpADE2) genes were sequentially disrupted, using an HpURA3 pop‐out cassette in H. polymorpha to generate a series of new multiple auxotrophic strains, including up to a quintuple auxotrophic strain. Unexpectedly, the HpTRP1 deletion mutants required additional tryptophan supplementation for their full growth, even on complex media such as YPD. Despite the clearly increased resistance to 5‐fluoroanthranilic acid of the HpTRP1 deletion mutants, the HpTRP1 blaster cassette does not appear to be usable as a counter‐selection marker in H. polymorpha. Expression vectors carrying HpADE2, HpTRP1 or HpMET2 with their own promoters and terminators as selectable markers were constructed and used to co‐transform the quintuple auxotrophic strain for the targeted expression of a heterologous gene, Aspergillus saitoi MsdS, at the ER, the Golgi and the cell surface, respectively. The nucleotide sequences presented here were submitted to GenBank under Accession Nos AY795576 (HpTRP1), FJ226453 (HpMET2) and FJ493241 (HpADE2), respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

Molecular characterization of the SEC1 gene of Saccharomyces cerevisiae: Subcellular distribution of a protein required for yeast protein secretion

Strains of Saccharomyces cerevisiae harbouring temperature-sensitive mutations in the SEC1 and SEC5 genes exhibit an accumulation of post-Golgi secretory vesicles at 37°C. We have cloned a fragment of yeast DNA which carries two distinct genes, one of which complements a sec1 mutation, and the other a sec5 mutation. Genetic tests confirm that the sec1-complementing gene is indeed SEC1, and is essential for cell growth. Nucleotide sequence analysis reveals that the cloned SEC1 gene is the same as a previously sequenced sec1-complementing gene. The SEC1 sequence encodes a protein of 724 amino acids with a predicted molecular mass of 83 kDa. Antibodies purified from a polyclonal antiserum raised against the protein product of the cloned gene recognize a yeast protein of apparent molecular mass 78 kDa which is found in a detergent-resistant association with a rapidly sedimenting yeast subcellular fraction, behaviour which is suggestive of an interaction with a component of the yeast cytoskeleton.     

N-glycosylation by transfer of GlcNAc2 from dolichol-PP-GlcNAc2 to the protein moiety of the major yeast exoglucanase

Transfer of truncated oligosaccharides to yeast exoglucanase (Exg) in Saccharomyces cerevisiae alg1 has been investigated. When incubated at the non-permissive temperature, alg1 cells secreted into the culture medium, in addition to the exoglucanase glycoforms secreted by wild type, underglycosylated forms as well as material with ionic properties of the non-glycosylated enzyme. As expected, none of the latter had affinity towards concanavalin A, but part of it bound to wheat germ agglutinin (WGA), suggesting that it contained, in addition to non-glycosylated Exg, glycoforms carrying non-reducing terminal GlcNAc. Only the WGA-bound material could be labelled with galactosyltransferase; furthermore, the label could be released by treatment with peptide-N⁴-N-acetyl-β-glucosamine asparagine amidase. These results unambiguously demonstrate that GlcNAc₂ can be transferred from dolichol-PP-GlcNAc₂ to one or both sequons of yeast Exg. Accordingly, they support previous observations suggesting that this early intermediate is able to translocate in vivo in order to make its sugar portion accessible to the oligosaccharyltransferase in the lumen of the endoplasmic reticulum. © 1998 John Wiley & Sons, Ltd.     

Isolation and characterization of the TRP1 gene from the yeast Yarrowia lipolytica and multiple gene disruption using a TRP blaster

The TRP1 gene encoding N-(5'-phosphoribosyl)-anthranilate isomerase was isolated from the yeast Yarrowia lipolytica, in which only a few genetic marker genes are available. The Y. lipolytica TRP1 gene (YlTRP1) cloned by complementation of Y. lipolytica trp1 mutation was found to be a functional homologue of Saccharomyces cerevisiae TRP1. Since YlTRP1 could be used for counterselection in medium containing 5-fluoroanthranilic acid (5-FAA), we constructed TRP blasters that contained YlTRP1 flanked by a direct repeat of a sequence and allowed the recycling of the YlTRP1 marker. Using the TRP blasters the sequential disruption of target genes could be carried out within the same strain of Y. lipolytica. The nucleotide sequence of the YlTRP1 gene has been deposited at GenBank under Accession No. AF420590.