scholarly journals Novel CDC34 (UBC3) ubiquitin-conjugating enzyme mutants obtained by charge-to-alanine scanning mutagenesis.

1995 ◽  
Vol 15 (3) ◽  
pp. 1210-1219 ◽  
Author(s):  
Z W Pitluk ◽  
M McDonough ◽  
P Sangan ◽  
D K Gonda
Keyword(s):  
Cell Cycle ◽  
Cold Sensitivity ◽  
Null Mutant ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Mutant Proteins ◽  
Terminal Domain ◽  
Copy Numbers ◽  
Conjugating Enzyme

CDC34 (UBC3) encodes a ubiquitin-conjugating (E2) enzyme required for transition from the G1 phase to the S phase of the budding yeast cell cycle. CDC34 consists of a 170-residue catalytic N-terminal domain onto which is appended an acidic C-terminal domain. A portable determinant of cell cycle function resides in the C-terminal domain, but determinants for specific function must reside in the N-terminal domain as well. We have explored the utility of "charge-to-alanine" scanning mutagenesis to identify novel N-terminal domain mutants of CDC34 that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitination but that nevertheless are defective with respect to its cell cycle function. Such mutants may reveal determinants of specific in vivo function, such as those required for interaction with substrates or trans-acting regulators of activity and substrate selectivity. Three of 18 "single-scan" mutants (in which small clusters of charged residues were mutated to alanine) were compromised with respect to in vivo function. One mutant (cdc34-109, 111, 113A) targeted a 12-residue segment of the Cdc34 protein not found in most other E2s and was unable to complement a cdc34 null mutant at low copy numbers but could complement a null mutant when overexpressed from an induced GAL1 promoter. Combining adjacent pairs of single-scan mutants to produce "double-scan" mutants yielded four additional mutants, two of which showed heat and cold sensitivity conditional defects. Most of the mutant proteins expressed in Escheria coli displayed unfacilitated (E3-independent) ubiquitin-conjugating activity, but two mutants differed from wild-type and other mutant Cdc34 proteins in the extent of multiubiquitination they catalyzed during an autoubiquitination reation-conjugating enzyme function and have identified additional mutant alleles of CDC34 that will be valuable in further genetic and biochemical studies of Cdc34-dependent ubiquitination.

scholarly journals Flagellar Morphogenesis: Protein Targeting and Assembly in the Paraflagellar Rod of Trypanosomes

1999 ◽  
Vol 19 (12) ◽  
pp. 8191-8200 ◽  
Author(s):  
Philippe Bastin ◽  
Thomas H. MacRae ◽  
Susan B. Francis ◽  
Keith R. Matthews ◽  
Keith Gull
Keyword(s):  
Cell Cycle ◽  
Trypanosoma Brucei ◽  
Protein Targeting ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Reporter ◽  
Mutant Proteins ◽  
Green Fluorescent ◽  
A Minor ◽  
Fluorescent Protein Reporter

ABSTRACT The paraflagellar rod (PFR) of the African trypanosomeTrypanosoma brucei represents an excellent model to study flagellum assembly. The PFR is an intraflagellar structure present alongside the axoneme and is composed of two major proteins, PFRA and PFRC. By inducible expression of a functional epitope-tagged PFRA protein, we have been able to monitor PFR assembly in vivo. As T. brucei cells progress through their cell cycle, they possess both an old and a new flagellum. The induction of expression of tagged PFRA in trypanosomes growing a new flagellum provided an excellent marker of newly synthesized subunits. This procedure showed two different sites of addition: a major, polar site at the distal tip of the flagellum and a minor, nonpolar site along the length of the partially assembled PFR. Moreover, we have observed turnover of epitope-tagged PFRA in old flagella that takes place throughout the length of the PFR structure. Expression of truncated PFRA mutant proteins identified a sequence necessary for flagellum localization by import or binding. This sequence was not sufficient to confer full flagellum localization to a green fluorescent protein reporter. A second sequence, necessary for the addition of PFRA protein to the distal tip, was also identified. In the absence of this sequence, the mutant PFRA proteins were localized both in the cytosol and in the flagellum where they could still be added along the length of the PFR. This seven-amino-acid sequence is conserved in all PFRA and PFRC proteins and shows homology to a sequence in the flagellar dynein heavy chain of Chlamydomonas reinhardtii.

The C-terminal domain of Saccharomyces cerevisiae DNA topoisomerase II

1994 ◽  
Vol 14 (5) ◽  
pp. 3197-3207
Author(s):  
P R Caron ◽  
P Watt ◽  
J C Wang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Localization ◽  
Topoisomerase Ii ◽  
Dna Topoisomerase Ii ◽  
Dna Topoisomerase ◽  
Mutant Proteins ◽  
Terminal Domain ◽  
Catalytically Active

A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperature-dependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results.

Mutations at sites involved in Suc1 binding inactivate Cdc2

1991 ◽  
Vol 11 (12) ◽  
pp. 6177-6184
Author(s):  
B Ducommun ◽  
P Brambilla ◽  
G Draetta
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Specific Interaction ◽  
Physiological Role ◽  
Negative Effects ◽  
Alanine Scanning Mutagenesis ◽  
Cycle Progression ◽  
Mutant Proteins ◽  
Additional Protein

suc1+ encodes an essential cell cycle regulator of the fission yeast Schizosaccharomyces pombe. Its product, a 13-kDa protein, interacts with the Cdc2 protein kinase. Both positive and negative effects on cell cycle progression have been attributed to Suc1. To date, the exact mechanisms and the physiological role of the interaction between Suc1 and Cdc2 remain unclear. Here we have studied the molecular basis of this association. We show that Cdc2 can bind Suc1 or its mammalian homolog directly in the absence of any additional protein component. Using an alanine scanning mutagenesis method, we analyzed the interaction between Cdc2 and Suc1. We show that the integrity of several domains on the Cdc2 protein, including sites directly involved in catalytic activity, is required for binding to Suc1. Furthermore, Cdc2 mutant proteins unable to bind Suc1 (but able to bind cyclins) are nonfunctional when overexpressed in S. pombe, indicating that a specific interaction with Suc1 is required for Cdc2 function.

scholarly journals Translational Attenuation Mechanism of ErmB Induction by Erythromycin Is Dependent on Two Leader Peptides

2021 ◽  
Vol 12 ◽  
Author(s):  
Shasha Wang ◽  
Kai Jiang ◽  
Xinyue Du ◽  
Yanli Lu ◽  
Lijun Liao ◽  
...  
Keyword(s):  
Premature Termination ◽  
Regulatory Region ◽  
Terminal Region ◽  
Leader Peptide ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Major Mechanism ◽  
Attenuation Mechanism

Ribosome stalling on ermBL at the tenth codon (Asp) is believed to be a major mechanism of ermB induction by erythromycin (Ery). In this study, we demonstrated that the mechanism of ermB induction by Ery depends not only on ermBL expression but also on previously unreported ermBL2 expression. Introducing premature termination codons in ermBL, we proved that translation of the N-terminal region of ermBL is the key component for ermB induced by Ery, whereas translation of the C-terminal region of ermBL did not affect Ery-induced ermB. Mutation of the tenth codon (Asp10) of ermBL with other amino acids showed that the degree of induction in vivo was not completely consistent with the data from the in vitro toe printing assay. Alanine-scanning mutagenesis of ermBL demonstrated that both N-terminal residues (R7-K11) and the latter part of ermBL (K20-K27) are critical for Ery induction of ermB. The frameshifting reporter plasmid showed that a new leader peptide, ermBL2, exists in the ermB regulatory region. Further, introducing premature termination mutation and alanine-scanning mutagenesis of ermBL2 demonstrated that the N-terminus of ermBL2 is essential for induction by Ery. Therefore, the detailed function of ermBL2 requires further study.

scholarly journals HupB Is a Bacterial Nucleoid-Associated Protein with an Indispensable Eukaryotic-Like Tail

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Joanna Hołówka ◽  
Damian Trojanowski ◽  
Katarzyna Ginda ◽  
Bartosz Wojtaś ◽  
Bartłomiej Gielniewski ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mycobacterium Tuberculosis ◽  
Drug Development ◽  
Health Problem ◽  
Biological Function ◽  
Multidrug Resistant ◽  
Chromosome Organization ◽  
Terminal Domain ◽  
Associated Proteins

ABSTRACT In bacteria, chromosomal DNA must be efficiently compacted to fit inside the small cell compartment while remaining available for the proteins involved in replication, segregation, and transcription. Among the nucleoid-associated proteins (NAPs) responsible for maintaining this highly organized and yet dynamic chromosome structure, the HU protein is one of the most conserved and highly abundant. HupB, a homologue of HU, was recently identified in mycobacteria. This intriguing mycobacterial NAP is composed of two domains: an N-terminal domain that resembles bacterial HU, and a long and distinctive C-terminal domain that contains several PAKK/KAAK motifs, which are characteristic of the H1/H5 family of eukaryotic histones. In this study, we analyzed the in vivo binding of HupB on the chromosome scale. By using PALM (photoactivated localization microscopy) and ChIP-Seq (chromatin immunoprecipitation followed by deep sequencing), we observed that the C-terminal domain is indispensable for the association of HupB with the nucleoid. Strikingly, the in vivo binding of HupB displayed a bias from the origin (oriC) to the terminus (ter) of the mycobacterial chromosome (numbers of binding sites decreased toward ter). We hypothesized that this binding mode reflects a role for HupB in organizing newly replicated oriC regions. Thus, HupB may be involved in coordinating replication with chromosome segregation. IMPORTANCE We currently know little about the organization of the mycobacterial chromosome and its dynamics during the cell cycle. Among the mycobacterial nucleoid-associated proteins (NAPs) responsible for chromosome organization and dynamics, HupB is one of the most intriguing. It contains a long and distinctive C-terminal domain that harbors several PAKK/KAAK motifs, which are characteristic of the eukaryotic histone H1/H5 proteins. The HupB protein is also known to be crucial for the survival of tubercle bacilli during infection. Here, we provide in vivo experimental evidence showing that the C-terminal domain of HupB is crucial for its DNA binding. Our results suggest that HupB may be involved in organizing newly replicated regions and could help coordinate chromosome replication with segregation. Given that tuberculosis (TB) remains a serious worldwide health problem (10.4 million new TB cases were diagnosed in 2015, according to WHO) and new multidrug-resistant Mycobacterium tuberculosis strains are continually emerging, further studies of the biological function of HupB are needed to determine if this protein could be a prospect for novel antimicrobial drug development. IMPORTANCE We currently know little about the organization of the mycobacterial chromosome and its dynamics during the cell cycle. Among the mycobacterial nucleoid-associated proteins (NAPs) responsible for chromosome organization and dynamics, HupB is one of the most intriguing. It contains a long and distinctive C-terminal domain that harbors several PAKK/KAAK motifs, which are characteristic of the eukaryotic histone H1/H5 proteins. The HupB protein is also known to be crucial for the survival of tubercle bacilli during infection. Here, we provide in vivo experimental evidence showing that the C-terminal domain of HupB is crucial for its DNA binding. Our results suggest that HupB may be involved in organizing newly replicated regions and could help coordinate chromosome replication with segregation. Given that tuberculosis (TB) remains a serious worldwide health problem (10.4 million new TB cases were diagnosed in 2015, according to WHO) and new multidrug-resistant Mycobacterium tuberculosis strains are continually emerging, further studies of the biological function of HupB are needed to determine if this protein could be a prospect for novel antimicrobial drug development.

scholarly journals Residues Required for Bacillus subtilis PhoP DNA Binding or RNA Polymerase Interaction: Alanine Scanning of PhoP Effector Domain Transactivation Loop and α Helix 3

2004 ◽  
Vol 186 (5) ◽  
pp. 1493-1502 ◽  
Author(s):  
Yinghua Chen ◽  
Wael R. Abdel-Fattah ◽  
F. Marion Hulett
Keyword(s):  
Bacillus Subtilis ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Transcription Activation ◽  
Phosphate Deficiency ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Α Helix

ABSTRACT Bacillus subtilis PhoP is a member of the OmpR family of response regulators that activates or represses genes of the Pho regulon upon phosphorylation by PhoR in response to phosphate deficiency. Because PhoP binds DNA and is a dimer in solution independent of its phosphorylation state, phosphorylation of PhoP may optimize DNA binding or the interaction with RNA polymerase. We describe alanine scanning mutagenesis of the PhoP α loop and α helix 3 region of PhoPC (Val190 to E214) and functional analysis of the mutated proteins. Eight residues important for DNA binding were clustered between Val202 and Arg210. Using in vivo and in vitro functional analyses, we identified three classes of mutated proteins. Class I proteins (PhoPI206A, PhoPR210A, PhoPL209A, and PhoPH208A) were phosphorylation proficient and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Class II proteins (PhoPH205A and PhoPV204A) were phosphorylation proficient and could dimerize but could not bind DNA prior to phosphorylation. Members of this class had higher transcription activation in vitro than in vivo. The class III mutants, PhoPV202A and PhoPD203A, had a reduced rate of phosphotransfer and could dimerize but could not bind DNA or activate transcription in vivo or in vitro. Seven alanine substitutions in PhoP (PhoPV190A, PhoPW191A, PhoPY193A, PhoPF195A, PhoPG197A, PhoPT199A, and PhoPR200A) that specifically affected transcription activation were broadly distributed throughout the transactivation loop extending from Val190 to as far toward the C terminus as Arg200. PhoPW191A and PhoPR200A could not activate transcription, while the other five mutant proteins showed decreased transcription activation in vivo or in vitro or both. The mutagenesis studies may indicate that PhoP has a long transactivation loop and a short α helix 3, more similar to OmpR than to PhoB of Escherichia coli.

scholarly journals Regulation of p53 Localization and Activity by Ubc13

2006 ◽  
Vol 26 (23) ◽  
pp. 8901-8913 ◽  
Author(s):  
Aaron Laine ◽  
Ivan Topisirovic ◽  
Dayong Zhai ◽  
John C. Reed ◽  
Katherine L. B. Borden ◽  
...  
Keyword(s):  
Dna Damage ◽  
Transcriptional Activity ◽  
Ubiquitin Ligases ◽  
Wild Type ◽  
Terminal Domain ◽  
Ubiquitin Conjugating Enzyme ◽  
P53 Stability ◽  
Conjugating Enzyme

ABSTRACT The abundance and activity of p53 are regulated largely by ubiquitin ligases. Here we demonstrate a previously undisclosed regulation of p53 localization and activity by Ubc13, an E2 ubiquitin-conjugating enzyme. While increasing p53 stability, Ubc13 decreases p53 transcriptional activity and increases its localization to the cytoplasm, changes that require its ubiquitin-conjugating activity. Ubc13 elicits K63-dependent ubiquitination of p53, which attenuates Hdm2-induced polyubiquitination of p53. Ubc13 association with p53 requires an intact C-terminal domain of p53 and is markedly stronger with a p53 mutant that cannot tetramerize. Expression of Ubc13 in vivo increases the pool of monomeric p53, indicating that Ubc13 affects tetramerization of p53. Significantly, wild-type but not mutant Ubc13 is associated with polysomes and enriches p53 within this fraction. In response to DNA damage, Ubc13 is no longer capable of facilitating p53 monomerization, in part due to a decrease in its own levels which is p53 dependent. Our findings point to a newly discerned mechanism important in the regulation of p53 organization, localization, and activity by Ubc13.

scholarly journals Arabidopsis RAD23B regulates pollen development by mediating degradation of KRP1

2020 ◽  
Vol 71 (14) ◽  
pp. 4010-4019
Author(s):  
Lan Li ◽  
Bin Li ◽  
Chong Xie ◽  
Teng Zhang ◽  
Cecilia Borassi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stress Responses ◽  
Pollen Development ◽  
26S Proteasome ◽  
Regulatory Proteins ◽  
Proteasome Inhibitor Mg132 ◽  
Null Mutant ◽  
Cell Cycle Protein ◽  
Microspore Stage

Abstract The ubiquitin (Ub)/26S proteasome system (UPS) plays a key role in plant growth, development, and survival by directing the turnover of numerous regulatory proteins. In the UPS, the ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains function as hubs for ubiquitin-mediated protein degradation. Radiation sensitive 23 (RAD23), which has been identified as a UBL/UBA protein, contributes to the progression of the cell cycle, stress responses, ER proteolysis, and DNA repair. Here, we report that pollen development is arrested at the microspore stage in a rad23b null mutant. We demonstrate that RAD23B can directly interact with KIP-related protein 1 (KRP1) through its UBL-UBA domains. In addition, plants overexpressing KRP1 have defects in pollen development, which is a phenotype similar to the rad23b mutant. RAD23B promotes the degradation of KRP1 in vivo, which is accumulated following treatment with the proteasome inhibitor MG132. Our results indicate that RAD23B plays an important in pollen development by controlling the turnover of the key cell cycle protein, KRP1.

scholarly journals Mutational Analysis of an Extracytoplasmic-Function Sigma Factor To Investigate Its Interactions with RNA Polymerase and DNA

2006 ◽  
Vol 188 (5) ◽  
pp. 1935-1942 ◽  
Author(s):  
Megan J. Wilson ◽  
Iain L. Lamont
Keyword(s):  
Rna Polymerase ◽  
Protein Function ◽  
Sigma Factor ◽  
Sigma Factors ◽  
Alanine Scanning Mutagenesis ◽  
Sequence Alignments ◽  
Mutant Proteins ◽  
Promoter Recognition ◽  
Promoter Dna

ABSTRACT The extracytoplasmic-function (ECF) family of sigma factors comprises a large group of proteins required for synthesis of a wide variety of extracytoplasmic products by bacteria. Residues important for core RNA polymerase (RNAP) binding, DNA melting, and promoter recognition have been identified in conserved regions 2 and 4.2 of primary sigma factors. Seventeen residues in region 2 and eight residues in region 4.2 of an ECF sigma factor, PvdS from Pseudomonas aeruginosa, were selected for alanine-scanning mutagenesis on the basis of sequence alignments with other sigma factors. Fourteen of the mutations in region 2 had a significant effect on protein function in an in vivo assay. Four proteins with alterations in regions 2.1 and 2.2 were purified as His-tagged fusions, and all showed a reduced affinity for core RNAP in vitro, consistent with a role in core binding. Region 2.3 and 2.4 mutant proteins retained the ability to bind core RNAP, but four mutants had reduced or no ability to cause core RNA polymerase to bind promoter DNA in a band-shift assay, identifying residues important for DNA binding. All mutations in region 4.2 reduced the activity of PvdS in vivo. Two of the region 4.2 mutant proteins were purified, and each showed a reduced ability to cause core RNA polymerase to bind to promoter DNA. The results show that some residues in PvdS have functions equivalent to those of corresponding residues in primary sigma factors; however, they also show that several residues not shared with primary sigma factors contribute to protein function.

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