Lucy Robinson | Louisiana State University Health Sciences Center - Shreveport (original) (raw)

Papers by Lucy Robinson

Journal of Biological Chemistry, 2004

The Yck2 protein is a plasma membrane-associated casein kinase 1 isoform that attaches to membran... more The Yck2 protein is a plasma membrane-associated casein kinase 1 isoform that attaches to membranes via palmitoylation of its C terminus. We have demonstrated that Yck2p traffics to the plasma membrane on secretory vesicles. Because Akr1p, the palmitoyl transferase for Yck2p, is located on Golgi membranes, it is likely that Yck2p first associates with Golgi membranes, and then is somehow recruited to budding plasma membrane-destined vesicles. We show here that residues 499-546 are sufficient for minimal Yck2p palmitoylation and plasma membrane localization. We previously described normal plasma membrane targeting of a Yck2p construct with the final five amino acids of Ras2p substituting for the final two Cys residues of Yck2p. This Yck2p variant no longer requires Akr1p for membrane association, but targets normally. We have generated the C-terminal deletions previously shown to affect Yck2p membrane association in this variant to determine which residues are important for targeting and/or modification. We find that all of the sequences previously identified as important for plasma membrane association are required only for Akr1p-dependent modification. Furthermore, palmitoylation is sufficient for specific association of Yck2p with secretory vesicles destined for the plasma membrane. Finally, both C-terminal Cys residues are palmitoylated, and dual acylation is required for efficient membrane association.

Mitochondrion, 2018

Mitochondrial DNA (mtDNA) double-strand break (DSB) repair is essential for maintaining mtDNA int... more Mitochondrial DNA (mtDNA) double-strand break (DSB) repair is essential for maintaining mtDNA integrity, but little is known about the proteins involved in mtDNA DSB repair. Here, we utilize Saccharomyces cerevisiae as a eukaryotic model to identify proteins involved in mtDNA DSB repair. We show that Mhr1, a protein known to possess homologous DNA pairing activity in vitro, binds to mtDNA DSBs in vivo, indicating its involvement in mtDNA DSB repair. Our data also indicate that Yku80, a protein previously implicated in mtDNA DSB repair, does not compete with Mhr1 for binding to mtDNA DSBs. In fact, C-terminally tagged Yku80 could not be detected in yeast mitochondrial extracts. Therefore, we conclude that Mhr1, but not Yku80, is a potential mtDNA DSB repair factor in yeast.

Nucleic acids research, Jan 26, 2017

The mechanism of mitochondrial DNA (mtDNA) replication in Saccharomyces cerevisiae is controversi... more The mechanism of mitochondrial DNA (mtDNA) replication in Saccharomyces cerevisiae is controversial. Evidence exists for double-strand break (DSB) mediated recombination-dependent replication at mitochondrial replication origin ori5 in hypersuppressive ρ- cells. However, it is not clear if this replication mode operates in ρ+ cells. To understand this, we targeted bacterial Ku (bKu), a DSB binding protein, to the mitochondria of ρ+ cells with the hypothesis that bKu would bind persistently to mtDNA DSBs, thereby preventing mtDNA replication or repair. Here, we show that mitochondrial-targeted bKu binds to ori5 and that inducible expression of bKu triggers petite formation preferentially in daughter cells. bKu expression also induces mtDNA depletion that eventually results in the formation of ρ0 cells. This data supports the idea that yeast mtDNA replication is initiated by a DSB and bKu inhibits mtDNA replication by binding to a DSB at ori5, preventing mtDNA segregation to daughter ...

The Faseb Journal, Apr 1, 2009

The Faseb Journal, Apr 1, 2007

Journal of cell science, 2000

The Yck1p and Yck2p casein kinase 1 isoforms in yeast are essential peripheral plasma membrane-as... more The Yck1p and Yck2p casein kinase 1 isoforms in yeast are essential peripheral plasma membrane-associated protein kinases with roles in endocytosis, cellular morphogenesis and cytokinesis. The membrane targeting of these cytoplasmically oriented protein kinases requires normal secretory pathway function, but specific targeting factors have not been identified. To learn more about Yckp targeting, we characterized mutations that cause synthetic lethality with impairment of Yck function. We report here that these include mutations in two gene products that function in protein trafficking. One of these is the previously described t-SNARE Tlg2p, which participates in recycling of proteins to the Golgi. The other is a previously uncharacterized protein, Rgp1p, which appears to have a similar function. Loss of either Tlg2p or Rgp1p causes inefficient localization of Yck2p, suggesting that its transport may be directed, in part, by a targeting factor that must be recycled back to the Golgi.

The EMBO journal, Jan 16, 1997

In Saccharomyces cerevisiae, the redundant YCK1 and YCK2 genes (Yeast Casein Kinase 1) are requir... more In Saccharomyces cerevisiae, the redundant YCK1 and YCK2 genes (Yeast Casein Kinase 1) are required for viability. We describe here the molecular analysis of four mutations that eliminate the requirement for Yck activity. These mutations alter proteins that resemble the four subunits of clathrin adaptors (APs), with highest sequence similarity to those of the recently identified AP-3 complex. The four yeast subunits are associated in a high-molecular-weight complex. These proteins have no essential function and are not redundant for function with other yeast AP-related proteins. Combination of suppressor mutations with a clathrin heavy chain mutation (chc1-ts) confers no synthetic growth defects. However, a yck(ts) mutation shows a strong synthetic growth defect with chc1-ts. Moreover, endocytosis of Ste3p is dramatically decreased in yck(ts) cells and is partially restored by the AP suppressor mutations. These results suggest that vesicle trafficking at the plasma membrane requires...

Molecular and cellular biology, 1993

Casein kinase I is an acidotropic protein kinase class that is widely distributed among eukaryoti... more Casein kinase I is an acidotropic protein kinase class that is widely distributed among eukaryotic cell types. In the yeast Saccharomyces cerevisiae, the casein kinase I isoform encoded by the gene pair YCK1 and YCK2 is a 60- to 62-kDa membrane-associated form. The Yck proteins perform functions essential for growth and division; either alone supports growth, but loss of function of both is lethal. We report here that casein kinase I-like activity is associated with a soluble Yck2-beta-galactosidase fusion protein in vitro and that thermolabile protein kinase activity is exhibited by a protein encoded by fusion of a temperature-sensitive yck2 allele with lacZ. Cells carrying the yck2-2ts allele arrest at restrictive temperature with multiple, elongated buds containing multiple nuclei. This phenotype suggests that the essential functions of the Yck proteins include roles in bud morphogenesis, possibly in control of cell growth polarity, and in cytokinesis or cell separation. Further,...

Methods in enzymology, 2002

... 661 USE OF GFP IN YEAST CELLS [39] [39] Use of Green Fluorescent Protein in Living Yeast Cell... more ... 661 USE OF GFP IN YEAST CELLS [39] [39] Use of Green Fluorescent Protein in Living Yeast Cells By KELLY TATCHELL and LUCY C. ROBINSON I ntroduction Although protein tags have been used for decades ... e BP Cormack, RH Valdivia, and S. Falkow, Gene 173, 33 (1996 ...

G3 (Bethesda, Md.), 2012

Ipl1/Aurora B is the catalytic subunit of a protein kinase complex required for chromosome segreg... more Ipl1/Aurora B is the catalytic subunit of a protein kinase complex required for chromosome segregation and nuclear division. Before anaphase, Ipl1 is required to establish proper kinetochore-microtubule associations and to regulate the spindle assembly checkpoint (SAC). The phosphatase Glc7/PP1 opposes Ipl1 for these activities. To investigate Ipl1 and Glc7 regulation in more detail, we isolated and characterized mutations in the yeast Saccharomyces cerevisiae that raise the restrictive temperature of the ipl-2 mutant. These suppressors include three intragenic, second-site revertants in IPL1; 17 mutations in Glc7 phosphatase components (GLC7, SDS22, YPI1); two mutations in SHP1, encoding a regulator of the AAA ATPase Cdc48; and a mutation in TCO89, encoding a subunit of the TOR Complex 1. Two revertants contain missense mutations in microtubule binding components of the kinetochore. rev76 contains the missense mutation duo1-S115F, which alters an essential component of the DAM1/DAS...

Yeast, 1987

CDC3, C'DC25 and CDC42 were localized to chromosome XI1 by hybridizing the cloned genes to Southe... more CDC3, C'DC25 and CDC42 were localized to chromosome XI1 by hybridizing the cloned genes to Southern blots of chromosomes separated by orthogonal-field-alternation gel electrophoresis. Meiotic tetrad analyses further localized these genes to the region distal to the RDNl locus on the right arm of the chromosome. The STEII gene, which had previously been mapped to chromosome XI1 (Chaleff and Tatchell, 1985), was found to be tightly linked to ZLV5. The data suggest a map order of CEN12-RDNI-CDC42-(CDC25-CDC3)-(ILV5-STEII)-URA4. Certain oddities of the data set raise the possibility that there may be constraints on the patterns of recombination in this region ofchromosome XII. KEY WORDS-cell cycle genes; genetic mapping; Saccharomyces; OFAGE. INTRODUCTTON The Saccharomyces cerevisiae CDC3 and CDC42 genes are involved in the morphogenetic processes of the cell cycle (Hartwell, 1971; Pringle and Hartwell, 1981; A. Adams, C. Evans, B. Sloat and J. Pringle, manuscript in preparation). The CDC25 gene appears to be involved in the control of growth via the RAS/cAMP/protein kinase system (Pringle and Hartwell, 1981; Robinson et al., 1987). All three genes were identified using temperature-sensitive (rs) lethal mutations and have since been isolated from yeast genomic-DNA libraries by plasmid complementation of these mutations (Robinson er al., 1987, and references cited therein; B. Haarer, D. Johnson and J. Pringle, unpublished results). However, these genes have heretofore evaded genetic mapping, despite several efforts using a variety of techniques. Recent advances in the separation of large DNA molecules using orthogonal-field-alternation gel electrophoresis (OFAGE) (Schwartz and Cantor,

Science, 1985

At a TA of 0C, however, the wing-beat frequency of the temperature-conforming geometrids should b... more At a TA of 0C, however, the wing-beat frequency of the temperature-conforming geometrids should be decreased considerably. If the geometrid wing-beat frequency is halved at 0C, it would be approximately 30 times lower than that of the noctuids. Geometrids and other nocturnal nonfeeding Lepidoptera generally have large wings (1) relative to their mass (low wing loading), and this characteristic confers a low energetic cost of flight (25). The mean wing-loading in Eupsilia sp. was 43 mg/cm2 (24). In the winter-flying Operophtera and Alsophila, however, wingloading (3.20 mg/cm2 and 3.90 mg/cm2) is not only 10 to 14 times lower than in the noctuids but is also lower than in all other geometrid (and other moth) species from both tropical (1, 26) and temperate regions (2) that have been examined. Low wing-loading that decreases the cost of transport may be a preadaptation in these geometrids that has allowed them to fly at low ambient and muscle temperatures. Our findings suggest that although morphology affecting thermoregulation can vary radically, the adaptations at the enzyme level for temperature are apparently highly conservative.

Science, 1987

The yeast Saceharomyces cere'ia contains two functional homologues of the ras oncogene family, RA... more The yeast Saceharomyces cere'ia contains two functional homologues of the ras oncogene family, RASI and RAS2. These genes are required for growth, and all

Proceedings of the National Academy of Sciences, 1985

Saccharomyces cerevisiae contains two genes with remarkable homology to members of the ras oncoge... more Saccharomyces cerevisiae contains two genes with remarkable homology to members of the ras oncogene family. These two genes, RAS] and RAS2, constitute an essential gene family since spores with disruptions ofboth genes fail to grow. We report here that strains containing RAS2 disruptions have three distinct phenotypes. First, they fail to grow efficiently on nonfermentable carbon sources. Second, they hyperaccumulate the storage carbohydrates glycogen and trehalose. Third, diploid cells homozygous for the RAS2 disruptions sporulate on rich media. Extragenic suppressors have been isolated that suppress the gluconeogenic defect. These suppressors fall into at least three complementation groups, mutations in two of which bypass the normal requirement ofRAS for cell viability, allowing cells containing neither RAS gene to grow. The phenotype of the RAS2 mutant and extragenic suppressors unplicateRAS with some function in the normal response to nutrient limitation.

Proceedings of the National Academy of Sciences, 2011

Results Reduced TORC1 Activity Suppresses the Temperature Sensitivity of ipl1-2. ipl1-2 strains g... more Results Reduced TORC1 Activity Suppresses the Temperature Sensitivity of ipl1-2. ipl1-2 strains grow at wild-type rates at 24°C but fail to grow at 30°C and above. To identify ipl1-2 suppressors, we

MGG Molecular & General Genetics, 1991

The TFS1 gene of Saccharomyces cerevisiae is a dosage-dependent suppressor of cdc25 mutations. Ov... more The TFS1 gene of Saccharomyces cerevisiae is a dosage-dependent suppressor of cdc25 mutations. Overexpression of TFS1 does not alleviate defects of temperature-sensitive adenylyl cyclase (cdc35) or ras2 disruption mutations. The ability of TFS1 to suppress cdc25 is allele specific: the temperature-sensitive edc25-1 mutation is suppressed efficiently but the cdc25-5 mutation and two disruption mutations are only partially suppressed. TFS1 maps to a previously undefined locus on chromosome XII between RDN1 and CDC42. The DNA sequence of TFS1 contains a single long open reading frame encoding a 219 amino acid polypeptide that is similar in sequence to two mammalian brain proteins. Insertion and deletion mutations in TFS1 are haploviable, indicating that TFS1 is not essential for growth.