Light-dependent N-end rule-mediated disruption of protein function in Saccharomyces cerevisiae and Drosophila melanogaster

Here we describe the development and characterization of the photo-N-degron, a peptide tag that can be used in optogenetic studies of protein function in vivo. The photo-N-degron can be expressed as a genetic fusion to the amino termini of other proteins, where it undergoes a blue light-dependent conformational change that exposes a signal for the class of ubiquitin ligases, the N-recognins, which mediate the N-end rule mechanism of proteasomal degradation. We demonstrate that the photo-N-degron can be used to direct light-mediated degradation of proteins in Saccharomyces cerevisiae and Drosophila melanogaster with fine temporal control. In addition, we compare the effectiveness of the photo-N-degron with that of two other light-dependent degrons that have been developed in their abilities to mediate the loss of function of Cactus, a component of the dorsal-ventral patterning system in the Drosophila embryo. We find that like the photo-N-degron, the blue light-inducible degradation (B-LID) domain, a light-activated degron that must be placed at the carboxy terminus of targeted proteins, is also effective in eliciting light-dependent loss of Cactus function, as determined by embryonic dorsal-ventral patterning phenotypes. In contrast, another previously described photosensitive degron (psd), which also must be located at the carboxy terminus of associated proteins, has little effect on Cactus-dependent phenotypes in response to illumination of developing embryos. These and other observations indicate that care must be taken in the selection and application of light-dependent and other inducible degrons for use in studies of protein function in vivo, but importantly demonstrate that N- and C-terminal fusions to the photo-N-degron and the B-LID domain, respectively, support light-dependent degradation in vivo.     

Saccharomyces cerevisiae homoserine kinase is homologous to prokaryotic homoserine kinases

The Saccharomyces cerevisiae gene (THR1) encoding homoserine kinase (HK; EC 2.7.1.39) was cloned by complementation in yeast. Disruption of the THR1 gene results in threonine auxotrophy in yeast. Comparison of the amino acid sequences of yeast and bacterial HKs reveals substantial similarity.     

Identification of novel filament-forming proteins in Saccharomyces cerevisiae and Drosophila melanogaster

The discovery of large supramolecular complexes such as the purinosome suggests that subcellular organization is central to enzyme regulation. A screen of the yeast GFP strain collection to identify proteins that assemble into visible structures identified four novel filament systems comprised of glutamate synthase, guanosine diphosphate–mannose pyrophosphorylase, cytidine triphosphate (CTP) synthase, or subunits of the eIF2/2B translation factor complex. Recruitment of CTP synthase to filaments and foci can be modulated by mutations and regulatory ligands that alter enzyme activity, arguing that the assembly of these structures is related to control of CTP synthase activity. CTP synthase filaments are evolutionarily conserved and are restricted to axons in neurons. This spatial regulation suggests that these filaments have additional functions separate from the regulation of enzyme activity. The identification of four novel filaments greatly expands the number of known intracellular filament networks and has broad implications for our understanding of how cells organize biochemical activities in the cytoplasm.     

Behavior of a Drosophila melanogaster transposable element in Saccharomyces cerevisiae.

The Drosophila melanogaster transposable element 412 is transiently unstable in Saccharomyces cerevisiae when present on a freely replicating plasmid. The 412 element undergoes recombination to form two circular molecules, a 412 deletion plasmid and, presumably, a 412 circle. The 412 deletion plasmid contains a single long terminal repeat which most likely is the result of homologous recombination within the long terminal repeats. This recombination occurs at or shortly after transformation and is independent of both the RAD52 gene product and the Flp gene of 2 micron DNA.     

An essential Saccharomyces cerevisiae single-stranded DNA binding protein is homologous to the large subunit of human RP-A.

Single-stranded DNA binding proteins (SSBs) are known to play a role in DNA replication and recombination in prokaryotes. An SSB was previously purified from the yeast Saccharomyces cerevisiae. This SSB stimulated the activity of a cognate strand exchange protein (SEP1) in vitro suggesting a role in recombination. We have cloned and functionally analyzed the gene encoding this protein. DNA sequencing of the cloned DNA revealed a 621 amino acid open reading frame with a coding potential for a Mr 70,269 polypeptide. Highly significant amino acid homology was detected between this S.cerevisiae gene and the Mr 70,000 subunit polypeptide of human RP-A, a cellular protein essential for SV40 DNA replication in vitro. Therefore, we named the S.cerevisiae gene RPA1. RPA1 encodes an essential function in this organism as shown by tetrad analysis of heterozygous insertion mutants and is continuously required for mitotic growth. Cells lacking RPA1 accumulate as multiply budded cells with a single nucleus suggesting a defect in DNA replication.     

The ADE2 gene from Saccharomyces cerevisiae: sequence and new vectors

We have determined the sequence of a DNA fragment encoding the ADE2 gene from Saccharomyces cerevisiae. A DNA fragment of 2241 bp capable of complementing ade2 mutations was modified so it is available as a single BglII fragment for use in yeast vectors or for gene disruptions. The minimal fragment codes for a putative protein which is highly similar to the protein encoded by the ADE6 gene from Schizosaccharomyces pombe and to the proteins encoded by the purEK operon of Escherichia coli.     

Hybridoma antibodies as specific probes to Drosophila melanogaster yolk polypeptides

Hybridoma antibodies to Drosophila melanogaster soluble yolk proteins (YPs) were developed by both in vivo and in vitro immunizations followed by the fusion of SP2/0-Ag14 cells and splenocytes of BALB/c mice. Rabbit antiserum was made female specific by affinity column with male proteins as ligand. The binding sites of these hybridoma antibodies and rabbit antibodies towards different YP components were identified with a combination of gel electrophoresis, Western blotting and immunohistochemical staining. A double antibody sandwich enzyme-linked immunosorbent assay was developed with monoclonal antibodies from 2 cell lines and alkaline phosphatase labelled rabbit polyclonal antibodies as primary and secondary antibodies respectively. Yolk polypeptide levels in the haemolymph can be monitored in individual insect samples.     

A Drosophila melanogaster gene encodes a protein homologous to the mouse t complex polypeptide 1

We have isolated and sequenced a cDNA from Drosophila melanogaster that is homologous to the mouse Tcp-1 gene encoding the t complex polypeptide 1, TCP-1. The Drosophila gene maps by in situ hybridization to bands 94B1-2 of the polytene chromosomes. It shares 66% nucleotide sequence identity with the mouse gene. The predicted Drosophila protein consists of 557 amino acids and shares 72% identity with the mouse polypeptide. The TCP-1 polypeptide appears to be highly conserved in evolution from mammals to simple eukaryotes because the Drosophila gene probe also detects related sequences in DNA from the yeast, Saccharomyces cerevisiae. The presence of TCP-1-related polypeptides in organisms such as Drosophila and yeast should facilitate biochemical and genetic analysis of its function.     

The Drosophila melanogaster brainiac protein is a glycolipid-specific beta 1,3N-acetylglucosaminyltransferase

Mutations at the Drosophila melanogaster brainiac locus lead to defective formation of the follicular epithelium during oogenesis and to neural hyperplasia. The brainiac gene encodes a type II transmembrane protein structurally similar to mammalian beta1,3-glycosyltransferases. We have cloned the brainiac gene from D. melanogaster genomic DNA and expressed it as a FLAG-tagged recombinant protein in Sf9 insect cells. Glycosyltransferase assays showed that brainiac is capable of transferring N-acetylglucosamine (GlcNAc) to beta-linked mannose (Man), with a marked preference for the disaccharide Man(beta1,4)Glc, the core of arthro-series glycolipids. The activity of brainiac toward arthro-series glycolipids was confirmed by showing that the enzyme efficiently utilized glycolipids from insects as acceptors whereas it did not with glycolipids from mammalian cells. Methylation analysis of the brainiac reaction product revealed a beta1,3 linkage between GlcNAc and Man, proving that brainiac is a beta1,3GlcNAc-transferase. Human beta1,3GlcNAc-transferases structurally related to brainiac were unable to transfer GlcNAc to Man(beta1,4)Glc-based acceptor substrates and failed to rescue a homozygous lethal brainiac allele, indicating that these proteins are paralogous and not orthologous to brainiac.     

Preliminary characterisation of DML1, an essential Saccharomyces cerevisiae gene related to misato of Drosophila melanogaster

A genetic and cell-biological analysis is provided for Saccharomyces cerevisiae DML1 (YMR211w) encoding a Drosophila melanogaster Misato-like protein. Misato and Dml1p are descendants of an ancestral tubulin-like protein, and exhibit regions with similarity to members of a GTPase family that include eukaryotic tubulin and prokaryotic FtsZ. Deletion of DML1 was lethal to haploid cells; sporulated DML1/dml1Delta heterozygotes from different genetic backgrounds gave rise to no more than two viable spores per tetrad. DAPI staining for DNA in combination with Southern analysis using the mitochondrial genes COX3, 15S_rRNA_2, and COB revealed that a significant portion of the surviving meiotic progeny were [rho(0)] lacking mtDNA. In addition, meiotic transmission of centromeric plasmids also appeared to be impaired. Self-complementation using extra-chromosomal copies of DML1 efficiently restored meiotic inheritance of mtDNA, but improved spore viability ratios only in part. Inheritance of mtDNA could also be restored using misato cDNA. Unscheduled expression of DML1 tethered to the inducible ADH2 promoter altered both mitochondrial dispersion and general cell morphology. We propose that Dml1p and Misato have been co-opted into a role in mtDNA inheritance in yeast, and into a cell division-related mechanism in flies, respectively. Dml1p might additionally function in the partitioning of the mitochondrial organelle itself, or in the segregation of chromosomes, thereby explaining its essential requirement.     

Cloning and characterization of the homologue of the Saccharomyces cerevisiae gene in Drosophila melanogaster]

RAD23 is an evolutionary conserved protein, which is essential for DNA excision repair. It is believed that this protein is present in all eukaryotic organisms from yeast to mammals. In this work, molecular cloning of the Drosophila melanogaster RAD23 gene and an analysis of the encoded protein are reported.     

Ribosomal protein L2 in Saccharomyces cerevisiae is homologous to ribosomal protein L1 in Xenopus laevis. Isolation and characterization of the genes

By cross-hybridization with a cDNA probe for the Xenopus laevis ribosomal protein L1 we have been able to isolate the homologous genes from a Saccharomyces cerevisiae genomic library. We have shown that these genes code for a ribosomal protein which was previously named L2. In yeast, like in X. laevis, these genes are present in two copies per haploid genome and, unlike the vertebrate counterpart, they do not contain introns. Amino acid comparison of the X. laevis L1 and S. cerevisiae L2 proteins has shown the presence of a highly conserved protein domain embedded in very divergent sequences. Although these sequences are very poorly homologous, they confer an overall secondary structure and folding highly conserved in the two species.