Perturbation of the Nucleus: A Novel Hog1p-independent, Pkc1p-dependent Consequence of Hypertonic Shock in Yeast

Hypertonic shock of Saccharomyces cerevisiae activates the Hog1p MAP kinase cascade. In contrast, protein kinase C (Pkc1p) and the “cell integrity” MAP kinase cascade are critical for the response to hypotonic shock. We observed that hypertonic shock transiently relocated many, but not all, nuclear and nucleolar proteins to the cytoplasm. We hypothesized that the relocation of nuclear proteins was due to activation of the Hog1p kinase cascade, yet, surprisingly, Hog1p was not required for these effects. In contrast, Pkc1p kinase activity was required, although the Pkc1p MAP kinase cascade and several factors known to lie upstream and downstream of Pkc1p were not. Moreover, sudden induction of a hyperactive form of Pkc1p was sufficient to relocate nuclear proteins. Taken together, these observations show that the scope of involvement of Pkc1p in the organization of the nucleus considerably exceeds what has been characterized previously. The relocation of nuclear proteins is likely to account for the profound inhibition of RNA synthesis that was observed during hypertonic shock.

Yeast Gga Coat Proteins Function with Clathrin in Golgi to Endosome Transport

Gga proteins represent a newly recognized, evolutionarily conserved protein family with homology to the “ear” domain of the clathrin adaptor AP-1 γ subunit. Yeast cells contain two Gga proteins, Gga1p and Gga2p, that have been proposed to act in transport between the trans-Golgi network and endosomes. Here we provide genetic and physical evidence that yeast Gga proteins function in trans-Golgi network clathrin coats. Deletion of Gga2p (gga2Δ), the major Gga protein, accentuates growth and α-factor maturation defects in cells carrying a temperature-sensitive allele of the clathrin heavy chain gene. Cells carrying either gga2Δ or a deletion of the AP-1 β subunit gene (apl2Δ) alone are phenotypically normal, but cells carrying both gga2Δ and apl2Δ are defective in growth, α-factor maturation, and transport of carboxypeptidase S to the vacuole. Disruption of both GGA genes and APL2 results in cells so severely compromised in growth that they form only microcolonies. Gga proteins can bind clathrin in vitro and cofractionate with clathrin-coated vesicles. Our results indicate that yeast Gga proteins play an important role in cargo-selective clathrin-mediated protein traffic from the trans-Golgi network to endosomes.

Conserved promoter elements in the CYP6B gene family suggest common ancestry for cytochrome P450 monooxygenases mediating furanocoumarin detoxification.

Despite the fact that Papilio glaucus and Papilio polyxenes share no single hostplant species, both species feed to varying extents on hostplants that contain furanocoumarins. P. glaucus contains two nearly identical genes, CYP6B4v2 and CYP6B5v1, and P. polyxenes contains two related genes, CYP6B1v3 and CYP6B3v2. Except for CYP6B3v2, the substrate specificity of which has not yet been defined, each of the encoded cytochrome P450 monooxygenases (P450s) metabolizes an array of linear furanocoumarins. All four genes are transcriptionally induced in larvae by exposure to the furanocoumarin xanthotoxin; several are also induced by other furanocoumarins. Comparisons of the organizational structures of these genes indicate that all have the same intron/exon arrangement. Sequences in the promoter regions of the P. glaucus CYP6B4v2/CYP6B5v1 genes and the P. polyxenes CYP6B3v2 gene are similar but not identical to the -146 to -97 region of CYP6B1v3 gene, which contains a xanthotoxin-responsive element (XRE-xan) important for basal and xanthotoxin-inducible transcription of CYP6B1v3. Complements of the xenobiotic-responsive element (XRE-AhR) in the dioxin-inducible human and rat CYP1A1 genes also exist in all four promoters, suggesting that these genes may be regulated by dioxin. Antioxidant-responsive elements (AREs) in mouse and rat glutathione S-transferase genes and the Barbie box element (Bar) in the bacterial CYP102 gene exist in the CYP6B1v3, CYP6B4v2, and CYP6B5v1 promoters. Similarities in the protein sequences, intron positions, and xanthotoxin- and xenobiotic-responsive promoter elements indicate that these insect CYP6B genes are derived from a common ancestral gene. Evolutionary comparisons between these P450 genes are the first available for a group of insect genes transcriptionally regulated by hostplant allelochemicals and provide insights into the process by which insects evolve specialized feeding habits.

DNA strand annealing is promoted by the yeast Rad52 protein.

The Saccharomyces cerevisiae RAD52 gene plays a pivotal role in genetic recombination. Here we demonstrate that yeast Rad52 is a DNA binding protein. To show that the interaction between Rad52 and DNA is direct and not mediated by other yeast proteins and to facilitate protein purification, a recombinant expression system was developed. The recombinant protein can bind both single- and double-stranded DNA and the addition of either Mg2+ or ATP does not enhance the binding of single-stranded DNA. Furthermore, a DNA binding domain was found in the evolutionary conserved N terminus of the protein. More importantly, we show that the protein stimulates DNA annealing even in the presence of a large excess of nonhomologous DNA. Rad52-promoted annealing follows second-order kinetics and the rate is 3500-fold faster than that of the spontaneous reaction. How this annealing activity relates to the genetic phenotype associated with rad52 mutant cells is discussed.

Mammalian orthologues of a yeast regulator of nonsense transcript stability.

All eukaryotes that have been studied to date possess the ability to detect and degrade transcripts that contain a premature signal for the termination of translation. This process of nonsense-mediated RNA decay has been most comprehensively studied in the yeast Saccharomyces cerevisiae where at least three trans-acting factors (Upf1p through Upf3P) are required. We have cloned cDNAs encoding human and murine homologues of Upf1p, termed rent1 (regulator of nonsense transcripts). Rent1 is the first identified mammalian protein that contains all of the putative functional elements in Upf1p including zinc finger-like and NTPase domains, as well as all motifs common to members of helicase superfamily I. Moreover, expression of a chimeric protein, N and C termini of Upf1p, complements the Upf1p-deficient phenotype in yeast. Thus, despite apparent differences between yeast and mammalian nonsense-mediated RNA decay, these data suggest that the two pathways use functionally related machinery.

Split invertase polypeptides form functional complexes in the yeast periplasm in vivo.

The assembly of functional proteins from fragments in vivo has been recently described for several proteins, including the secreted maltose binding protein in Escherichia coli. Here we demonstrate for the first time that split gene products can function within the eukaryotic secretory system. Saccharomyces cerevisiae strains able to use sucrose produce the enzyme invertase, which is targeted by a signal peptide to the central secretory pathway and the periplasmic space. Using this enzyme as a model we find the following: (i) Polypeptide fragments of invertase, each containing a signal peptide, are independently translocated into the endoplasmic reticulum (ER) are modified by glycosylation, and travel the entire secretory pathway reaching the yeast periplasm. (ii) Simultaneous expression of independently translated and translocated overlapping fragments of invertase leads to the formation of an enzymatically active complex, whereas individually expressed fragments exhibit no activity. (iii) An active invertase complex is assembled in the ER, is targeted to the yeast periplasm, and is biologically functional, as judged by its ability to facilitate growth on sucrose as a single carbon source. These observation are discussed in relation to protein folding and assembly in the ER and to the trafficking of proteins through the secretory pathway.

The methylotrophic yeast Pichia pastoris synthesizes a functionally active chromophore precursor of the plant photoreceptor phytochrome.

Induction of the expression of an algal phytochrome cDNA in the methylotrophic yeast Pichia pastoris led to time-dependent formation of photoactive holophytochrome without the addition of exogenous bilins. Both in vivo and in vitro difference spectra of this phytochromic species are very similar to those of higher plant phytochrome A, supporting the conclusion that this species possesses a phytochromobilin prosthetic group. Zinc blot analyses confirm that a bilin chromophore is covalently bound to the algal phytochrome apoprotein. The hypothesis that P. pastoris contains phytochromobilin synthase, the enzyme that converts biliverdin IX alpha to phytochromobilin, was also addressed in this study. Soluble extracts from P. pastoris were able to convert biliverdin to a bilin pigment, which produced a native difference spectrum upon assembly with oat apophytochrome A. HPLC analyses confirm that biliverdin is converted to both 3E- and 3Z-isomers of phytochromobilin. These investigations demonstrate that the ability to synthesize phytochromobilin is not restricted to photosynthetic organisms and support the hypothesis of a more widespread distribution of the phytochrome photoreceptor.

Mammalian Sug1 and c-Fos in the nuclear 26S proteasome.

In a search for regulatory proteins that interact with the leucine zipper motif of c-Fos in the yeast two-hybrid screen, we have identified a protein (FZA-B) that has extensive sequence similarity to SUG1 of Saccharomyces cerevisiae. Here we show that FZA-B can functionally substitute for SUG1 in yeast and that FZA-B interacts with Fos proteins in vitro through their leucine zippers. In rat liver and in HeLa cells, FZA-B is present in the 26S proteasome complex, as is c-Fos. Immobilized antibody raised against an FZA-B-specific peptide depleted peptidase activity, proteasomal proteins, FZA-B, and c-Fos from a 26S proteasome preparation. FZA-B is found predominantly in the nuclear fraction of COS cells expressing an FZA-B transgene and in the nuclear 26S proteasome of HeLa cells. We conclude that FZA-B is the mammalian homolog of SUG1 (mSug1) and that it is present in the nuclear 26S proteasome of cells. Our results suggest that mSug1 may be involved in the degradation of c-Fos and other transcription factors.

Association of yeast SIN1 with the tetratrico peptide repeats of CDC23.

The yeast SIN1 protein is a nuclear protein that together with other proteins behaves as a transcriptional repressor of a family of genes. In addition, sin1 mutants are defective in proper mitotic chromosome segregation. In an effort to understand the basis for these phenotypes, we employed the yeast two-hybrid system to identify proteins that interact with SIN1 in vivo. Here we demonstrate that CDC23, a protein known to be involved in sister chromatid separation during mitosis, is able to directly interact with SIN1. Furthermore, using recombinant molecules in vitro, we show that the N terminal of SIN1 is sufficient to bind a portion of CDC23 consisting solely of tetratrico peptide repeats. Earlier experiments identified the C-terminal domain of SIN1 to be responsible for interaction with a protein that binds the regulatory region of HO, a gene whose transcription is repressed by SIN1. Taken together with the results presented here, we suggest that SIN1 is a chromatin protein having at least a dual function: The N terminal of SIN1 interacts with the tetratrico peptide repeat domains of CDC23, a protein involved in chromosome segregation, whereas the C terminal of SIN1 binds proteins involved in transcriptional regulation.

Exon shuffling and the origin of the mitochondrial targeting function in plant cytochrome c1 precursor.

Since most of the examples of "exon shuffling" are between vertebrate genes, the view is often expressed that exon shuffling is limited to the evolutionarily recent lineage of vertebrates. Although exon shuffling in plants has been inferred from the analysis of intron phases of plant genes [Long, M., Rosenberg, C. & Gilbert, W. (1995) Proc. Natl. Acad. Sci. USA 92, 12495-12499] and from the comparison of two functionally unknown sunflower genes [Domon, C. & Steinmetz, A. (1994) Mol. Gen. Genet. 244, 312-317], clear cases of exon shuffling in plant genes remain to be uncovered. Here, we report an example of exon shuffling in two important nucleus-encoded plant genes: cytosolic glyceraldehyde-3-phosphate dehydrogenase (cytosolic GAPDH or GapC) and cytochrome c1 precursor. The intron-exon structures of the shuffled region indicate that the shuffling event took place at the DNA sequence level. In this case, we can establish a donor-recipient relationship for the exon shuffling. Three amino terminal exons of GapC have been donated to cytochrome c1, where, in a new protein environment, they serve as a source of the mitochondrial targeting function. This finding throws light upon an old important but unsolved question in gene evolution: the origin of presequences or transit peptides that generally exist in nucleus-encoded organelle genes.

Ferulate-5-hydroxylase from Arabidopsis thaliana defines a new family of cytochrome P450-dependent monooxygenases.

The fah1 mutant of Arabidopsis is defective in the accumulation of sinapic acid-derived metabolites, including the guaiacyl-syringyl lignin typical of angiosperms. Earlier results indicated that the FAH1 locus encodes ferulate-5-hydroxylase (F5H), a cytochrome P450-dependent monooxygenase (P450) of the general phenylpropanoid pathway. We have cloned the gene encoding this P450 by T-DNA tagging and have confirmed the identity of the cloned gene by complementation of the mutant phenotype. F5H shows 34% amino acid sequence identity with the avocado ripening-induced P450 CYP71A1 and 32% identity with the flavonoid-3',5'-hydroxylases of Petunia hybrida. In contrast, it shares much less homology with cinnamate-4-hydroxylase, a P450 that catalyzes the hydroxylation of cinnamic acid three steps earlier in the general phenylpropanoid pathway. Since the highest degree of identity between F5H and previously sequenced P450s is only 34%, F5H identifies a new P450 subfamily that has been designated CYP84.

The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins.

Although transcription and pre-mRNA processing are colocalized in eukaryotic nuclei, molecules linking these processes have not previously been described. We have identified four novel rat proteins by their ability to interact with the repetitive C-terminal domain (CTD) of RNA polymerase II in a yeast two-hybrid assay. A yeast homolog of one of the rat proteins has also been shown to interact with the CTD. These CTD-binding proteins are all similar to the SR (serine/arginine-rich) family of proteins that have been shown to be involved in constitutive and regulated splicing. In addition to alternating Ser-Arg domains, these proteins each contain discrete N-terminal or C-terminal CTD-binding domains. We have identified SR-related proteins in a complex that can be immunoprecipitated from nuclear extracts with antibodies directed against RNA polymerase II. In addition, in vitro splicing is inhibited either by an antibody directed against the CTD or by wild-type but not mutant CTD peptides. Thus, these results suggest that the CTD and a set of CTD-binding proteins may act to physically and functionally link transcription and pre-mRNA processing.

In vitro reconstitution of human replication factor C from its five subunits.

Replication factor C (RFC, also called Activator I) is part of the processive eukaryotic DNA polymerase holoenzymes. The processive elongation of DNA chains requires that DNA polymerases are tethered to template DNA at primer ends. In eukaryotes the ring-shaped homotrimeric protein, proliferating cell nuclear antigen (PCNA), ensures tight template-polymerase interaction by encircling the DNA strand. Proliferating cell nuclear antigen is loaded onto DNA through the action of RFC in an ATP-dependent reaction. Human RFC is a protein complex consisting of five distinct subunits that migrate through SDS/polyacrylamide gels as protein bands of 140, 40, 38, 37, and 36 kDa. All five genes encoding the RFC subunits have been cloned and sequenced. A functionally identical RFC complex has been isolated from Saccharomyces cerevisiae and the deduced amino acid sequences among the corresponding human and yeast subunits are homologous. Here we report the expression of the five cloned human genes using an in vitro coupled transcription/translation system and show that the gene products form a complex resembling native RFC that is active in supporting an RFC-dependent replication reaction. Studies on the interactions between the five subunits suggest a cooperative mechanism in the assembly of the RFC complex. A three-subunit core complex, consisting of p36, p37, and p40, was identified and evidence is presented that p38 is essential for the interaction between this core complex and the large p140 subunit.

Effect of deletion of 5'HS3 or 5'HS2 of the human beta-globin locus control region on the developmental regulation of globin gene expression in beta-globin locus yeast artificial chromosome transgenic mice.

To analyze the function of the 5' DNase I hypersensitive sites (HSs) of the locus control region (LCR) on beta-like globin gene expression, a 2.3-kb deletion of 5'HS3 or a 1.9-kb deletion of 5'HS2 was recombined into a beta-globin locus yeast artificial chromosome, and transgenic mice were produced. Deletion of 5'HS3 resulted in a significant decrease of epsilon-globin gene expression and an increase of gamma-globin gene expression in embryonic cells. Deletion of 5'HS2 resulted in only a small decrease in expression of epsilon-, gamma-, and beta-globin mRNA at all stages of development. Neither deletion affected the temporal pattern of globin gene switching. These results suggest that the LCR contains functionally redundant elements and that LCR complex formation does not require the presence of all DNase I hypersensitive sites. The phenotype of the 5'HS3 deletion suggests that individual HSs may influence the interaction of the LCR with specific globin gene promoters during the course of ontogeny.

The human multidrug resistance-associated protein functionally complements the yeast cadmium resistance factor 1.

A Saccharomyces cerevisiae strain with a disrupted yeast cadmium resistance factor (YCF1) gene (DTY168) is hypersensitive to cadmium. YCF1 resembles the human multidrug resistance-associated protein MRP (63% amino acid similarity), which confers resistance to various cytotoxic drugs by lowering the intracellular drug concentration. Whereas the mechanism of action of YCF1 is not known, MRP was recently found to transport glutathione S-conjugates across membranes. Here we show that expression of the human MRP cDNA in yeast mutant DTY168 cells restores cadmium resistance to the wild-type level. Transport of S-(2,4-dinitrobenzene)-glutathione into isolated yeast microsomal vesicles is strongly reduced in the DTY168 mutant and this transport is restored to wild-type level in mutant cells expressing MRP cDNA. We find in cell fractionation experiments that YCF1 is mainly localized in the vacuolar membrane in yeast, whereas MRP is associated both with the vacuolar membrane and with other internal membranes in the transformed yeast cells. Our results indicate that yeast YCF1 is a glutathione S-conjugate pump, like MRP, and they raise the possibility that the cadmium resistance in yeast involves cotransport of cadmium with glutathione derivatives.