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

PLoS Genetics ◽  
2021 ◽  
Vol 17 (5) ◽  
pp. e1009544
Author(s):  
Leslie M. Stevens ◽  
Goheun Kim ◽  
Theodora Koromila ◽  
John W. Steele ◽  
James McGehee ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drosophila Melanogaster ◽  
Blue Light ◽  
Protein Function ◽  
Loss Of Function ◽  
Carboxy Terminus ◽  
Direct Light ◽  
Associated Proteins ◽  
Lid Domain

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.

La proteins from Drosophila melanogaster and Saccharomyces cerevisiae: a yeast homolog of the La autoantigen is dispensable for growth

1994 ◽  
Vol 14 (8) ◽  
pp. 5412-5424
Author(s):  
C J Yoo ◽  
S L Wolin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drosophila Melanogaster ◽  
Protein Function ◽  
Rna Polymerase Iii ◽  
Biochemical Properties ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Polymerase Iii ◽  
La Protein

The human autoantigen La is a 50-kDa protein which binds to the 3' termini of virtually all nascent polymerase III transcripts. Experiments with mammalian transcription extracts have led to the proposal that the La protein is required for multiple rounds of transcription by RNA polymerase III (E. Gottlieb and J. A. Steitz, EMBO J. 8:851-861, 1989; R. J. Maraia, D. J. Kenan, and J. D. Keene, Mol. Cell. Biol. 14:2147-2158, 1994). Although La protein homologs have been identified in a variety of vertebrate species, the protein has not been identified in invertebrates. In order to begin a genetic analysis of La protein function, we have characterized homologs of the La protein in the fruit fly Drosophila melanogaster and the yeast Saccharomyces cerevisiae. We show that both the Drosophila and yeast La proteins are bound to precursors of polymerase III RNAs in vivo. The Drosophila and yeast proteins resemble the human La protein in their biochemical properties, as both proteins can be partially purified from cells by a procedure previously devised to purify the human protein. Similarly to vertebrate La proteins, the Drosophila and yeast homologs preferentially bind RNAs that terminate with a 3' hydroxyl. Despite the fact that the La protein is conserved between humans and Saccharomyces cerevisiae, yeast cells containing a null allele of the gene encoding the La protein are viable, suggesting that another protein(s) plays a functionally redundant role.

scholarly journals La proteins from Drosophila melanogaster and Saccharomyces cerevisiae: a yeast homolog of the La autoantigen is dispensable for growth.

1994 ◽  
Vol 14 (8) ◽  
pp. 5412-5424 ◽  
Author(s):  
C J Yoo ◽  
S L Wolin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drosophila Melanogaster ◽  
Protein Function ◽  
Rna Polymerase Iii ◽  
Biochemical Properties ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Polymerase Iii ◽  
La Protein

The human autoantigen La is a 50-kDa protein which binds to the 3' termini of virtually all nascent polymerase III transcripts. Experiments with mammalian transcription extracts have led to the proposal that the La protein is required for multiple rounds of transcription by RNA polymerase III (E. Gottlieb and J. A. Steitz, EMBO J. 8:851-861, 1989; R. J. Maraia, D. J. Kenan, and J. D. Keene, Mol. Cell. Biol. 14:2147-2158, 1994). Although La protein homologs have been identified in a variety of vertebrate species, the protein has not been identified in invertebrates. In order to begin a genetic analysis of La protein function, we have characterized homologs of the La protein in the fruit fly Drosophila melanogaster and the yeast Saccharomyces cerevisiae. We show that both the Drosophila and yeast La proteins are bound to precursors of polymerase III RNAs in vivo. The Drosophila and yeast proteins resemble the human La protein in their biochemical properties, as both proteins can be partially purified from cells by a procedure previously devised to purify the human protein. Similarly to vertebrate La proteins, the Drosophila and yeast homologs preferentially bind RNAs that terminate with a 3' hydroxyl. Despite the fact that the La protein is conserved between humans and Saccharomyces cerevisiae, yeast cells containing a null allele of the gene encoding the La protein are viable, suggesting that another protein(s) plays a functionally redundant role.

scholarly journals Gemin3 Is an Essential Gene Required for Larval Motor Function and Pupation in Drosophila

2009 ◽  
Vol 20 (1) ◽  
pp. 90-101 ◽  
Author(s):  
Karl B. Shpargel ◽  
Kavita Praveen ◽  
T. K. Rajendra ◽  
A. Gregory Matera
Keyword(s):  
Protein Function ◽  
Muscular Atrophy ◽  
Essential Gene ◽  
Survival Motor Neuron ◽  
Loss Of Function ◽  
Transposon Insertion ◽  
Associated Proteins ◽  
Smn Protein

The assembly of metazoan Sm-class small nuclear ribonucleoproteins (snRNPs) is an elaborate, step-wise process that takes place in multiple subcellular compartments. The initial steps, including formation of the core RNP, are mediated by the survival motor neuron (SMN) protein complex. Loss-of-function mutations in human SMN1 result in a neuromuscular disease called spinal muscular atrophy. The SMN complex is comprised of SMN and a number of tightly associated proteins, collectively called Gemins. In this report, we identify and characterize the fruitfly ortholog of the DEAD box protein, Gemin3. Drosophila Gemin3 (dGem3) colocalizes and interacts with dSMN in vitro and in vivo. RNA interference for dGem3 codepletes dSMN and inhibits efficient Sm core assembly in vitro. Transposon insertion mutations in Gemin3 are larval lethals and also codeplete dSMN. Transgenic overexpression of dGem3 rescues lethality, but overexpression of dSMN does not, indicating that loss of dSMN is not the primary cause of death. Gemin3 mutant larvae exhibit motor defects similar to previously characterized Smn alleles. Remarkably, appreciable numbers of Gemin3 mutants (along with one previously undescribed Smn allele) survive as larvae for several weeks without pupating. Our results demonstrate the conservation of Gemin3 protein function in metazoan snRNP assembly and reveal that loss of either Smn or Gemin3 can contribute to neuromuscular dysfunction.

scholarly journals Cross-Species RNAi Rescue Platform in Drosophila melanogaster

Genetics ◽  
2009 ◽  
Vol 183 (3) ◽  
pp. 1165-1173 ◽  
Author(s):  
Shu Kondo ◽  
Matthew Booker ◽  
Norbert Perrimon
Keyword(s):  
Cell Culture ◽  
Drosophila Melanogaster ◽  
Large Scale ◽  
Transgenic Animals ◽  
Selection Marker ◽  
Bacterial Artificial Chromosomes ◽  
Loss Of Function ◽  
Artificial Chromosomes ◽  
Target Effects

RNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.

scholarly journals Conformational dynamics is key to understanding loss-of-function of NQO1 cancer-associated polymorphisms and its correction by pharmacological ligands

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Encarnación Medina-Carmona ◽  
Rogelio J. Palomino-Morales ◽  
Julian E. Fuchs ◽  
Esperanza Padín-Gonzalez ◽  
Noel Mesa-Torres ◽  
...  
Keyword(s):  
Protein Dynamics ◽  
Protein Function ◽  
Conformational Dynamics ◽  
Loss Of Function ◽  
Quinone Oxidoreductase ◽  
Protein Levels ◽  
Terminal Domain ◽  
Directional Preference

Abstract Protein dynamics is essential to understand protein function and stability, even though is rarely investigated as the origin of loss-of-function due to genetic variations. Here, we use biochemical, biophysical, cell and computational biology tools to study two loss-of-function and cancer-associated polymorphisms (p.R139W and p.P187S) in human NAD(P)H quinone oxidoreductase 1 (NQO1), a FAD-dependent enzyme which activates cancer pro-drugs and stabilizes several oncosuppressors. We show that p.P187S strongly destabilizes the NQO1 dimer in vitro and increases the flexibility of the C-terminal domain, while a combination of FAD and the inhibitor dicoumarol overcome these alterations. Additionally, changes in global stability due to polymorphisms and ligand binding are linked to the dynamics of the dimer interface, whereas the low activity and affinity for FAD in p.P187S is caused by increased fluctuations at the FAD binding site. Importantly, NQO1 steady-state protein levels in cell cultures correlate primarily with the dynamics of the C-terminal domain, supporting a directional preference in NQO1 proteasomal degradation and the use of ligands binding to this domain to stabilize p.P187S in vivo. In conclusion, protein dynamics are fundamental to understanding loss-of-function in p.P187S and to develop new pharmacological therapies to rescue this function.

In silico investigation of Methyl parathion and Diazinon with different Metabolic protein in Drosophila melanogaster

2021 ◽  
pp. 3794-3798
Author(s):  
Suman Sahoo ◽  
Md. Lutfur Rahman ◽  
Sagarika Mitra ◽  
Rajiniraja M.
Keyword(s):  
Drosophila Melanogaster ◽  
Protein Function ◽  
Methyl Parathion ◽  
Function Analysis ◽  
Glutathione S Transferase ◽  
Agriculture Industry ◽  
Bimolecular Interactions ◽  
Metabolic Protein ◽  
Chemical Pollutant

Chemical pollutant such as insecticide, pesticide and drugs are mainly used for agriculture, industry and economic development, which are well known for environment pollutant due to its toxicity and persistence in the nature. It can accumulate into the environment and continuously contaminate the food chain which causes threat to the health of consumer including human. Based on all these studies our investigation deals with the effects of two insecticides viz. methyl parathion and diazinon to non target organism like Drosophila melanogaster. In this study we have performed molecular modeling, docking and protein function analysis of different metabolic and physiological enzyme of Drosophila melanogaster such as acetylcholinesterase (AchE), Glutathione S-transferase D1(GST) and Protein kinase C (PKC) with these insecticides of six combinations (AchE + Diazinon, AchE + methyl parathion, GST+Diazinon, GST+Methyl parathion, PKC+Diazinon, PKC+Methyl parathion). Molecular docking results showing best binding affinity for GST+ Methyl parathion with binding energy of -4.79 kcal/mol. Overall, methyl parathion produces efficient binding toward all target protein when compare to diazinon. However, more detailed analysis need to be carried out to have an in-depth understanding of in vivo significance of these bimolecular interactions.

scholarly journals mRNAs Encoding Telomerase Components and Regulators Are Controlled by UPF Genes in Saccharomyces cerevisiae

2003 ◽  
Vol 2 (1) ◽  
pp. 134-142 ◽  
Author(s):  
Jeffrey N. Dahlseid ◽  
Jodi Lew-Smith ◽  
Michael J. Lelivelt ◽  
Shinichiro Enomoto ◽  
Amanda Ford ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomere Length ◽  
Dna Sequences ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Telomeric Silencing ◽  
Expression Of Genes ◽  
Mutant Strains ◽  
Genes Encoding

ABSTRACT Telomeres, the chromosome ends, are maintained by a balance of activities that erode and replace the terminal DNA sequences. Furthermore, telomere-proximal genes are often silenced in an epigenetic manner. In Saccharomyces cerevisiae, average telomere length and telomeric silencing are reduced by loss of function of UPF genes required in the nonsense-mediated mRNA decay (NMD) pathway. Because NMD controls the mRNA levels of several hundred wild-type genes, we tested the hypothesis that NMD affects the expression of genes important for telomere functions. In upf mutants, high-density oligonucleotide microarrays and Northern blots revealed that the levels of mRNAs were increased for genes encoding the telomerase catalytic subunit (Est2p), in vivo regulators of telomerase (Est1p, Est3p, Stn1p, and Ten1p), and proteins that affect telomeric chromatin structure (Sas2p and Orc5p). We investigated whether overexpressing these genes could mimic the telomere length and telomeric silencing phenotypes seen previously in upf mutant strains. Increased dosage of STN1, especially in combination with increased dosage of TEN1, resulted in reduced telomere length that was indistinguishable from that in upf mutants. Increased levels of STN1 together with EST2 resulted in reduced telomeric silencing like that of upf mutants. The half-life of STN1 mRNA was not altered in upf mutant strains, suggesting that an NMD-controlled transcription factor regulates the levels of STN1 mRNA. Together, these results suggest that NMD maintains the balance of gene products that control telomere length and telomeric silencing primarily by maintaining appropriate levels of STN1, TEN1, and EST2 mRNA.

scholarly journals The REG2 gene of Saccharomyces cerevisiae encodes a type 1 protein phosphatase-binding protein that functions with Reg1p and the Snf1 protein kinase to regulate growth.

1996 ◽  
Vol 16 (6) ◽  
pp. 2922-2931 ◽  
Author(s):  
D L Frederick ◽  
K Tatchell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Protein Phosphatase ◽  
Slow Growth ◽  
Glucose Repression ◽  
Growth Defect ◽  
Loss Of Function ◽  
Cell Extracts

The GLC7 gene of Saccharomyces cerevisiae encodes the catalytic subunit of type 1 protein phosphatase (PP1) and is essential for cell growth. We have isolated a previously uncharacterized gene, REG2, on the basis of its ability to interact with Glc7p in the two-hybrid system. Reg2p interacts with Glc7p in vivo, and epitope-tagged derivatives of Reg2p and Glc7p coimmunoprecipitate from cell extracts. The predicted protein product of the REG2 gene is similar to Reg1p, a protein believed to direct PP1 activity in the glucose repression pathway. Mutants with a deletion of reg1 display a mild slow-growth defect, while reg2 mutants exhibit a wild-type phenotype. However, mutants with deletions of both reg1 and reg2 exhibit a severe growth defect. Overexpression of REG2 complements the slow-growth defect of a reg1 mutant but does not complement defects in glycogen accumulation or glucose repression, two traits also associated with a reg1 deletion. These results indicate that REG1 has a unique role in the glucose repression pathway but acts together with REG2 to regulate some as yet uncharacterized function important for growth. The growth defect of a reg1 reg2 double mutant is alleviated by a loss-of-function mutation in the SNF1-encoded protein kinase. The snf1 mutation also suppresses the glucose repression defects of reg1. Together, our data are consistent with a model in which Reg1p and Reg2p control the activity of PP1 toward substrates that are phosphorylated by the Snf1p kinase.

scholarly journals End4p/Sla2p Interacts with Actin-associated Proteins for Endocytosis in Saccharomyces cerevisiae

1997 ◽  
Vol 8 (11) ◽  
pp. 2291-2306 ◽  
Author(s):  
A. Wesp ◽  
L. Hicke ◽  
J. Palecek ◽  
R. Lombardi ◽  
T. Aust ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Temperature ◽  
Mutational Analysis ◽  
Coiled Coil ◽  
Temperature Sensitive ◽  
Cortical Actin ◽  
Actin Organization ◽  
Coiled Coil Domain ◽  
Associated Proteins

end4–1 was isolated as a temperature-sensitive endocytosis mutant. We cloned and sequenced END4 and found that it is identical to SLA2/MOP2. This gene is required for growth at high temperature, viability in the absence of Abp1p, polarization of the cortical actin cytoskeleton, and endocytosis. We used a mutational analysis of END4 to correlate in vivo functions with regions of End4p and we found that two regions of End4p participate in endocytosis but that the talin-like domain of End4p is dispensable. The N-terminal domain of End4p is required for growth at high temperature, endocytosis, and actin organization. A central coiled-coil domain of End4p is necessary for formation of a soluble sedimentable complex. Furthermore, this domain has an endocytic function that is redundant with the function(s) ofABP1 and SRV2. The endocytic function of Abp1p depends on its SH3 domain. In addition we have isolated a recessive negative allele of SRV2 that is defective for endocytosis. Combined biochemical, functional, and genetic analysis lead us to propose that End4p may mediate endocytosis through interaction with other actin-associated proteins, perhaps Rvs167p, a protein essential for endocytosis.

scholarly journals In vivo expression and mitochondrial targeting of yeast apoiso-1-cytochrome c fusion proteins.

1990 ◽  
Vol 10 (11) ◽  
pp. 5753-5762 ◽  
Author(s):  
S H Nye ◽  
R C Scarpulla
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Fusion Proteins ◽  
Intermembrane Space ◽  
Heme Binding ◽  
Mitochondrial Targeting ◽  
Carboxy Terminus ◽  
Coding Region ◽  
Respiratory Growth

To define the import pathway for apoiso-1-cytochrome c in vivo, the coding region for bacterial chloramphenicol acetyltransferase (CAT) or yeast copper metallothionein (CuMT) was fused to the carboxy terminus of the apoiso-1-cytochrome c (iso-1) coding region. When the resulting iso-1/CAT and iso-1/CuMT fusion proteins were individually expressed in Saccharomyces cerevisiae, they were specifically targeted to the mitochondria and protected from trypsin digestion. Although iso-1/CAT was accessible to heme modification, it remained membrane associated because of the folded conformation of the CAT domain. A small deletion disrupting CAT structure resulted in the translocation of the resulting fusion protein, iso-1/CAT delta, to the intermembrane space, where it functioned efficiently in respiratory electron transfer. Similarly, iso-1/CuMT was heme modified and nearly identical to iso-1 in its ability to support respiratory growth, indicating that the CuMT domain was compatible with translocation to the IMS. Inclusion of copper in the growth medium, which converts the loosely structured apo-CuMT to a tightly folded holo-CuMT, inhibited both heme attachment and respiratory growth without affecting mitochondrial targeting. Thus, by altering the folded conformation of the reporter moiety of these fusion proteins, it was possible to differentiate between those molecules arrested at the mitochondrial targeting step of the cytochrome c import pathway and those translocated to the intermembrane space. By replacing the heme-binding cysteine residues with serines, this system was used to demonstrate that the import requirement for heme attachment operated at the level of membrane translocation and not on mitochondrial targeting in vivo.

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