Sls1p stimulates Sec63p-mediated activation of Kar2p in a conformation-dependent manner in the yeast endoplasmic reticulum

We previously characterized the SLS1 gene in the yeast Yarrowia lipolytica and showed that it interacts physically with YlKar2p to promote translocation across the endoplasmic-reticulum membrane (A. Boisramé, M. Kabani, J. M. Beckerich, E. Hartmann, and C. Gaillardin, J. Biol. Chem. 273:30903-30908, 1998). A Y. lipolytica Kar2p mutant was isolated that restored interaction with an Sls1p mutant, suggesting that the interaction with Sls1p could be nucleotide and/or conformation dependent. This result was used as a working hypothesis for more accurate investigations in Saccharomyces cerevisiae. We show by two-hybrid an in vitro assays that the S. cerevisiae homologue of Sls1p interacts with ScKar2p. Using dominant lethal mutants of ScKar2p, we were able to show that ScSls1p preferentially interacts with the ADP-bound conformation of the molecular chaperone. Synthetic lethality was observed between DeltaScsls1 and translocation-deficient kar2 or sec63-1 mutants, providing in vivo evidence for a role of ScSls1p in protein translocation. Synthetic lethality was also observed with ER-associated degradation and folding-deficient kar2 mutants, strongly suggesting that Sls1p functions are not restricted to the translocation process. We show that Sls1p stimulates in a dose-dependent manner the binding of ScKar2p on the lumenal J domain of Sec63p fused to glutathione S-transferase. Moreover, Sls1p is shown to promote the Sec63p-mediated activation of Kar2p's ATPase activity. Our data strongly suggest that Sls1p could be the first GrpE-like protein described in the endoplasmic reticulum.     

Genetic evidence for selective degradation of RNA polymerase subunits by the 20S proteasome in Saccharomyces cerevisiae.

scs32 was isolated as an extragenic suppressor of a temperature-sensitive (ts) mutation (rpo26-31) in the gene encoding Rpo26p, a subunit common to yeast nuclear RNA polymerases (RNAPs). rpo26-31 also confers inositol auxotrophy, inhibits the assembly of RNAPI and RNAPII and reduces the steady-state level of Rpo26p and the largest subunit of RNAPI (Rpo11p or A190p) and RNAPII (Rpo21p). rpo26-31p accumulated to wild-type levels in the scs32 strain; nevertheless, the amount of assembled RNAPII remained at a reduced level at high temperature. Hence, scs32 only partially suppressed the ts phenotype and was unable to suppress the Ino-phenotype of rpo26-31. SCS32 is identical to PUP3, which encodes a subunit of the yeast proteasome. scs32 was able to suppress the phenotype of other ts alleles of RPO26, all of which reduce the steady-state level of this subunit. However, scs32 was unable to suppress the ts phenotype of mutant alleles of RPO21, or result in accumulation of the unstable rpo21-4p. These observations suggest that the stability of non-functional or unassembled forms of Rpo26p and Rpo21p are regulated independently.     

Nucleotide exchange factor for the yeast Hsp70 molecular chaperone Ssa1p

We report on the identification of Fes1p (yBR101cp) as a cytosolic homologue of Sls1p, an endoplasmic reticulum (ER) protein previously shown to act as a nucleotide exchange factor for yeast BiP (M. Kabani, J.-M. Beckerich, and C. Gaillardin, Mol. Cell. Biol. 20:6923-6934, 2000). We found that Fes1p associates preferentially to the ADP-bound form of the cytosolic Hsp70 molecular chaperone Ssa1p and promotes nucleotide release. Fes1p activity was shown to be compartment and species specific since Sls1p and Escherichia coli GrpE could not substitute for Fes1p. Surprisingly, whereas Sls1p stimulated the ATPase activity of BiP in cooperation with luminal J proteins, Fes1p was shown to inhibit the Ydj1p-mediated activation of Ssa1p ATPase activity in steady-state and single-turnover assays. Disruption of FES1 in several wild-type backgrounds conferred a strong thermosensitive phenotype but partially rescued ydj1-151 thermosensitivity. The Delta fes1 strain was proficient for posttranslational protein translocation, as well as for the ER-associated degradation of two substrates. However, the Delta fes1 mutant showed increased cycloheximide sensitivity and a general translational defect, suggesting that Fes1p acts during protein translation, a process in which Ssa1p and Ydj1p are known to be involved. In support of this hypothesis, Fes1p was found to be associated with ribosomes.     

A mutation in the secretion pathway of the yeast Yarrowia lipolytica that displays synthetic lethality in combination with a mutation affecting the signal recognition particle.

In an attempt to identify proteins involved in the translocation step of protein secretion, a genetic screen was carried out in the yeast Yarrowia lipolytica. A conditional lethal mutant which has a defect in the 7S RNA of the signal recognition particle was mutagenized and screened for second-site mutations that specifically exacerbate its temperature sensitivity. This approach had previously allowed the characterization of an endoplasmic reticulum component, Sls1p, involved in protein translocation. A second mutation, sls2-1, was isolated that causes synthetic lethality when combined with the 7S RNA mutation. On its own, the sls2-1 mutation confers a temperature-sensitive growth phenotype. The secretory phenotype of the sls2 mutant consists in abnormal secretion of several polypeptides, and thus differs from the defect in secretory protein synthesis associated with the 7S RNA and sls1-1 mutations. Two new Y. lipolytica genes were identified which can relieve the growth defect of sls2-1 cells: SLS2 itself and SSL2, a multicopy suppressor of the temperature sensitivity of the sls2 mutant. The SLS2 gene encodes a polypeptide that can potentially be farnesylated and phosphorylated, and shares some homology with an S. cerevisiae protein of unknown function. Ssl2p resembles calmodulin-dependent serine/threonine protein kinases. These two proteins may interact to regulate protein sorting. Received: 9 June 1998 / Accepted: 10 February 1999     

A mutation in the secretion pathway of the yeast Yarrowia lipolytica that displays synthetic lethality in combination with a mutation affecting the signal recognition particle

In an attempt to identify proteins involved in the translocation step of protein secretion, a genetic screen was carried out in the yeast Yarrowia lipolytica. A?conditional lethal mutant which has a defect in the 7S RNA of the signal recognition particle was mutagenized and screened for second-site mutations that specifically exacerbate its temperature sensitivity. This approach had previously allowed the characterization of an endoplasmic reticulum component, Sls1p, involved in protein translocation. A second mutation, sls2-1, was isolated that causes synthetic lethality when combined with the 7S RNA mutation. On its own, the sls2-1 mutation confers a temperature-sensitive growth phenotype. The secretory phenotype of the sls2 mutant consists in abnormal secretion of several polypeptides, and thus differs from the defect in secretory protein synthesis associated with the 7S RNA and sls1-1 mutations. Two new Y. lipolytica genes were identified which can relieve the growth defect of sls2-1 cells: SLS2 itself and SSL2, a multicopy suppressor of the temperature sensitivity of the sls2 mutant. The SLS2 gene encodes a polypeptide that can potentially be farnesylated and phosphorylated, and shares some homology with an S. cerevisiae protein of unknown function. Ssl2p resembles calmodulin-dependent serine/threonine protein kinases. These two proteins may interact to regulate protein sorting.     

A suppressor of an RNA polymerase II mutation of Saccharomyces cerevisiae encodes a subunit common to RNA polymerases I, II, and III.

RNA polymerase II (RNAPII) is a complex multisubunit enzyme responsible for the synthesis of pre-mRNA in eucaryotes. The enzyme is made of two large subunits associated with at least eight smaller polypeptides, some of which are common to all three RNA polymerase species. We have initiated a genetic analysis of RNAPII by introducing mutations in RPO21, the gene encoding the largest subunit of RNAPII in Saccharomyces cerevisiae. We have used a yeast genomic library to isolate plasmids that can suppress a temperature-sensitive mutation in RPO21 (rpo21-4), with the goal of identifying gene products that interact with the largest subunit of RNAPII. We found that increased expression of wild-type RPO26, a single-copy, essential gene encoding a 155-amino-acid subunit common to RNAPI, RNAPII, and RNAPIII, suppressed the rpo21-4 temperature-sensitive mutation. Mutations were constructed in vitro that resulted in single amino acid changes in the carboxy-terminal portion of the RPO26 gene product. One temperature-sensitive mutation, as well as some mutations that did not by themselves generate a phenotype, were lethal in combination with rpo21-4. These results support the idea that the RPO26 and RPO21 gene products interact.     

Isolation and characterization of temperature-sensitive RNA polymerase II mutants of Saccharomyces cerevisiae.

Three independent, recessive, temperature-sensitive (Ts-) conditional lethal mutations in the largest subunit of Saccharomyces cerevisiae RNA polymerase II (RNAP II) have been isolated after replacement of a portion of the wild-type gene (RPO21) by a mutagenized fragment of the cloned gene. Measurements of cell growth, viability, and total RNA and protein synthesis showed that rpo21-1, rpo21-2, and rpo21-3 mutations caused a slow shutoff of RNAP II activity in cells shifted to the nonpermissive temperature (39 degrees C). Each mutant displayed a distinct phenotype, and one of the mutant enzymes (rpo21-1) was completely deficient in RNAP II activity in vitro. RNAP I and RNAP III in vitro activities were not affected. These results were consistent with the notion that the genetic lesions affect RNAP II assembly or holoenzyme stability. DNA sequencing revealed that in each case the mutations involved nonconservative amino acid substitutions, resulting in charge changes. The lesions harbored by all three rpo21 Ts- alleles lie in DNA sequence domains that are highly conserved among genes that encode the largest subunits of RNAP from a variety of eucaryotes; one mutation lies in a possible Zn2+ binding domain.     

Interactions of Liguleless1 and Liguleless2 Function during Ligule Induction in Maize

The maize ligule is an adaxial membranous structure on the leaf that develops at the boundary of the sheath and blade. The ligule and the associated auricle are dispensable structures, amenable to genetic manipulation. We present here a genetic analysis of liguleless1 (lg1) and liguleless2 (lg2), the two genes known to be uniquely necessary for ligule and auricle development. We show that both reference mutant alleles, lg1-R and lg2-R, are null alleles. The double mutant phenotype suggests that lg1 and lg2 act in the same pathway. Indeed, the dosage of a functional allele at either gene affects the null phenotype of the other. While lg1 function has previously been shown to be cell-autonomous, here we show that the lg2-R phenotype is cell-nonautonomous, suggesting lg1 and lg2 play different roles in the ligule-auricle induction mechanism. We present a model in which early lg2 function specifies the precise position where ligule and auricle will develop. Later lg2 function interacts with lg1 function (either directly or indirectly) to transmit and receive a make-ligule-make-auricle inductive signal.     

Abnormal anxiety-related behavior in serotonin transporter null mutant mice: the influence of genetic background

Serotonin transporter (5-HTT) null mutant mice provide a model system to study the role genetic variation in the 5-HTT plays in the regulation of emotion. Anxiety-like behaviors were assessed in 5-HTT null mutants with the mutation placed on either a B6 congenic or a 129S6 congenic background. Replicating previous findings, B6 congenic 5-HTT null mutants exhibited increased anxiety-like behavior and reduced exploratory locomotion on the light ↔ dark exploration and elevated plus-maze tests. In contrast, 129S6 congenic 5-HTT null mutant mice showed no phenotypic abnormalities on either test. 5-HTT null mutants on the 129S6 background showed reduced 5-HT_1A receptor binding (as measured by quantitative autoradiography) and reduced 5-HT_1A receptor function (as measured by 8-OH-DPAT-indcued hypothermia). These data confirm that the 5-HTT null mutation produced alterations in brain 5-HT function in mice on the 129S6 background, thereby discounting the possibility that the absence of an abnormal anxiety-like phenotype in these mice was due to a suppression of the mutation by 129 modifier genes. Anxiety-like behaviors in the light ↔ dark exploration and elevated plus-maze tests were significantly higher in 129S6 congenic +/+ mice as compared to B6 congenic +/+ mice. This suggests that high baseline anxiety-like behavior in the 129S6 strain might have precluded detection of the anxiety-like effects of the 5-HTT null mutation on this background. Present findings provide further evidence linking genetic variation in the 5-HTT to abnormalities in mood and anxiety. Furthermore, these data highlight the utility of conducting behavioral phenotyping of mutant mice on multiple genetic backgrounds.     

PKCdelta requires p53 for suppression of the transformed phenotype in human colon cancer cells

We have previously demonstrated that the delta isoform of Protein Kinase C (PKCdelta) acts as a tumor suppressor in HCT116 human colon cancer cells, and that p21(waf1/cip1) is an essential downstream effector of PKCdelta. Our data suggested that p53 might also be involved in the suppression of the neoplastic phenotype induced by PKCdelta. Here we show that homozygous knockout of p53 renders the HCT116 cell line unresponsive to PKCdelta overexpression. Whereas reconstitution of p53 alone did not modify the morphology and growth properties of HCT116/p53null cells, overexpression of both p53 and PKCdelta induced a number of alterations indicating suppression of the transformed phenotype. Interestingly, PKCdelta was ineffective when overexpressed in HT29 cells, a human colon cancer line characterized by the Arg273His dominant-negative mutation of p53. Thus, our data indicate that wild-type p53 is an essential effector of PKCdelta in human colon cancer cells.     

Multiple cellular consequences of isocitrate dehydrogenase isozyme dysfunction

To probe the functions of multiple forms of isocitrate dehydrogenase in Saccharomyces cerevisiae, mutants lacking three of the isozymes were constructed and analyzed. Results show that, while the mitochondrial NAD+-dependent enzyme, IDH (composed of Idh1p and Idh2p subunits) is not the major contributor to total isocitrate dehydrogenase activity under any growth condition, loss of IDH produces the most dramatic growth phenotypes. These include reduced growth in the absence of glutamate, as well as an increase in expression of Idp2p (the cytosolic NADP+-dependent enzyme) under some growth conditions. In this study, we have focused on another phenotype associated with loss of IDH, an elevated frequency of petite mutations indicating loss of functional mtDNA. Using mutant forms of IDH with altered active site residues, a correlation was observed between the high frequency of petite mutations and the loss of catalytic activity. Loss of Idp1p (the mitochondrial NADP+-dependent enzyme) and Idp2p contributes to the loss of functional mtDNA, but only in an IDH dysfunctional background. Surprisingly, overexpression of Idp1p, but not of Idp2p, was found to result in an elevated petite frequency independent of the functional state of IDH. This is the first phenotype associated with altered Idp1p. Finally, throughout this study we examined effects of loss of mitochondrial citrate synthase (Cit1p) on isocitrate dehydrogenase mutants, since defects in the CIT1 gene were previously shown to enhance growth of IDH dysfunctional strains on nonfermentable carbon sources. Loss of Cit1p was found to suppress the petite phenotype of strains lacking IDH, suggesting that these phenotypes may be linked.     

An enzyme in yeast mitochondria that catalyzes a step in branched-chain amino acid biosynthesis also functions in mitochondrial DNA stability.

The yeast mitochondrial high mobility group protein Abf2p is required, under certain growth conditions, for the maintenance of wild-type (rho+) mitochondrial DNA (mtDNA). We have identified a multicopy suppressor of the mtDNA instability phenotype of cells with a null allele of the ABF2 gene (delta abf2). The suppressor is a known gene, ILV5, encoding the mitochondrial protein, acetohydroxy acid reductoisomerase, which catalyzes a step in branched-chain amino acid biosynthesis. Efficient suppression occurs with just a 2- to 3-fold increase in ILV5 copy number. Moreover, in delta abf2 cells with a single copy of ILV5, changes in mtDNA stability correlate directly with changes in conditions that are known to affect ILV5 expression. Wild-type mtDNA is unstable in cells with an ILV5 null mutation (delta ilv5), leading to the production of mostly rho- petite mutants. The instability of rho+ mtDNA in delta ilv5 cells is not simply a consequence of a block in branched-chain amino acid biosynthesis, since mtDNA is stable in cells with a null allele of the ILV2 gene, which encodes another enzyme of that pathway. The most severe instability of rho+ mtDNA is observed in cells with null alleles of both ABF2 and ILV5. We suggest that ILV5 encodes a bifunctional protein required for branched-chain amino acid biosynthesis and for the maintenance of rho+ mtDNA.     

Genetic factors influence cataract formation in alpha 3 connexin knockout mice

Connexin alpha 3 (Cx46 or Gja3) gene targeted null mice developed lens nuclear cataracts shortly after birth. A large variance in the cataracts was observed in alpha 3 null sibs on a mixed 129SvJae X C57BL/6J F3 background. This suggested that the genetic background might influence the cataract phenotype. Therefore, we placed the alpha 3 null mutation into a 129SvJae background, and also backcrossed the mutation for six generations into 129SvJ and C57BL/6J backgrounds. While alpha 3 nulls on the two 129 backgrounds contained severe cataracts associated with gamma crystallin cleavage, alpha 3 nulls on the C57B16 background had far milder cataracts with no detectable gamma crystallin cleavage. These findings suggest that a genetic modifier exists that influences gamma crystallin stability, and that gamma crystallin breakdown is associated with severe nuclear cataracts.