LGG-1/GABARAP lipidation is dispensable for autophagy and development in C .elegans

2021 ◽  
Author(s):  
Romane Leboutet ◽  
C&eacuteline Largeau ◽  
Magali Prigent ◽  
Gr&eacutegoire Quinet ◽  
Manuel S Rodriguez ◽  
...  
Keyword(s):  
Essential Role ◽  
Phosphatidyl Ethanolamine ◽  
Essential Gene ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Autophagic Flux ◽  
High Plasticity ◽  
Post Translational Modifications ◽  
C Elegans

The ubiquitin-like proteins Atg8/LC3/GABARAP are required for multiple steps of autophagy such as initiation, cargo recognition and engulfment, vesicle closure and degradation. Most of LC3/GABARAP functions are considered dependent on their post-translational modifications and addressing to membranes through a conjugation to a lipid, the phosphatidyl-ethanolamine. Contrarily to mammals, C. elegans possesses single homologs of LC3 and GABARAP families, named LGG-2 and LGG-1. Using site directed mutagenesis, we inhibited the conjugation of LGG-1 to the autophagosomal membrane and generated mutants that express only cytosolic forms, either the precursor or the cleaved protein. LGG-1 is an essential gene for autophagy and development in C. elegans, but we discovered that its functions could be fully achieved independently of its localization to the membrane. This study reveals an essential role for the cleaved form of LGG-1 in autophagy but also in an autophagy independent embryonic function. Our data question the use of the lipidated GABARAP/LC3 as the main marker of autophagic flux and highlight the high plasticity of autophagy.

scholarly journals Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase

2016 ◽  
Vol 473 (11) ◽  
pp. 1605-1615 ◽  
Author(s):  
Sara R. Costa ◽  
Magdalena Marek ◽  
Kristian B. Axelsen ◽  
Lisa Theorin ◽  
Thomas G. Pomorski ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Β Subunit ◽  
Post Translational Modifications ◽  
Functional Roles ◽  
Evolutionarily Conserved ◽  
A Site ◽  
P Type ◽  
Cell Division Control

P-type ATPases of subfamily IV (P4-ATPases) constitute a major group of phospholipid flippases that form heteromeric complexes with members of the Cdc50 (cell division control 50) protein family. Some P4-ATPases interact specifically with only one β-subunit isoform, whereas others are promiscuous and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues, Asn181 and Asn231. Whereas mutation of Asn231 seems to have a small effect on P4-ATPase complex formation, mutation of evolutionarily conserved Asn181 disrupts interaction between the two subunits. Of the four cysteine residues located in the ALIS5 ectodomain, mutation of Cys86 and Cys107 compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys158 and Cys172 has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications in the β-subunit have different functional roles in different organisms, which may be related to the promiscuity of the P4-ATPase.

scholarly journals Disintegrin-like/cysteine-rich domains of the reprolysin HF3: Site-directed mutagenesis reveals essential role of specific residues

Biochimie ◽  
2011 ◽  
Vol 93 (2) ◽  
pp. 345-351 ◽  
Author(s):  
Milene C. Menezes ◽  
Ana Karina de Oliveira ◽  
Robson L. Melo ◽  
Mônica Lopes-Ferreira ◽  
Vanessa Rioli ◽  
...  
Keyword(s):  
Essential Role ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Integrating bioinformatics tools to investigate protein phosphorylation

2015 ◽  
Author(s):  
Dimitrios Vlachakis ◽  
Elena Bencurova ◽  
Mangesh Bhide ◽  
Sophia Kossida
Keyword(s):  
Mass Spectrometry ◽  
Western Blot ◽  
Protein Phosphorylation ◽  
Gel Electrophoresis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Phosphorylation Sites ◽  
Post Translational Modifications ◽  
Cellular Processes ◽  
Benchmark Study

Protein phosphorylation is one of the most important protein post-translational modifications and plays a role in numerous cellular processes including recognition, signaling and degradation. It can be studied experimentally by various methodologies, like employing western blot analysis, site-directed mutagenesis, 2 D gel electrophoresis, mass spectrometry etc. A number of in silico tools have also been developed in order to predict plausible phosphorylation sites in a given protein. In this review, we conducted a benchmark study including the leading protein phosphorylation prediction software, in an effort to determine which performs best. The first place was taken by GPS 2.2, having predicted all phosphorylation sites with a 83% fidelity while in second place came NetPhos 2.0 with 69%.

scholarly journals Experimental evidence for the essential role of the C-terminal residue in the strict aminopeptidase activity of the thiol aminopeptidase PepC, a bacterial bleomycin hydrolase

1997 ◽  
Vol 328 (2) ◽  
pp. 343-347 ◽  
Author(s):  
Luis MATA ◽  
Marta ERRA-PUJADA ◽  
Jean-Claude GRIPON ◽  
Michel-Yves MISTOU
Keyword(s):  
Essential Role ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Aminopeptidase Activity ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Terminal Residue ◽  
Bleomycin Hydrolase ◽  
Eukaryotic Organisms

PepCs isolated from lactic acid bacteria and bleomycin hydrolases of eukaryotic organisms are strict aminopeptidases which belong to the papain family of thiol peptidases. The structural basis of the enzymic specificity of the lactococcal PepC has been investigated by site-directed mutagenesis. The deletion of the C-terminal residue (Ala-435) abolished the aminopeptidase activity, whereas this deletion led to a new peptidase specificity. The enzymic properties of wild-type and mutant PepCs demonstrate that the terminal α-carboxy group plays a key role in the strict aminopeptidase activity.

scholarly journals Site-Directed Mutagenesis of the 19-Kilodalton Lipoprotein Antigen Reveals No Essential Role for the Protein in the Growth and Virulence of Mycobacterium intracellulare

1998 ◽  
Vol 66 (8) ◽  
pp. 3626-3634 ◽  
Author(s):  
Eshwar Mahenthiralingam ◽  
Britt-Inger Marklund ◽  
Lucy A. Brooks ◽  
Debbie A. Smith ◽  
Gregory J. Bancroft ◽  
...  
Keyword(s):  
Essential Role ◽  
Genetic Manipulation ◽  
Virulent Strain ◽  
Site Directed Mutagenesis ◽  
Infection Model ◽  
Directed Mutagenesis ◽  
Exact Nature ◽  
Mycobacterium Intracellulare

ABSTRACT The mycobacterial 19-kilodalton antigen (19Ag) is a highly expressed, surface-associated glycolipoprotein which is immunodominant in infected patients and has little homology with other known proteins. To investigate the pathogenic significance of the 19Ag, site-directed mutagenesis of the Mycobacterium intracellulare 19Ag gene was carried out by using a suicide vector-based strategy. Allelic replacement of the 19Ag gene of a mouse-avirulent M. intracellulare strain, 1403, was achieved by double-crossover homologous recombination with a gentamicin resistance gene-mutated allele. Unfortunately, an isogenic 19Ag was not achievable in the mouse-virulent strain, D673. However, a 19Ag mutant was successfully constructed in M. intracellulare FM1, a chemically mutagenized derivative of strain D673. FM1 was more amenable to genetic manipulation and susceptible to site-directed mutagenesis of the 19Ag gene yet retained the virulent phenotype of the parental strain. No deleterious effects of 19Ag gene mutation were observed during in vitro growth ofM. intracellulare. Virulence assessment of the isogenic 19Ag mutants in a mouse infection model demonstrated that the antigen plays no essential role in the growth of M. intracellularein vivo. Site-directed mutagenesis of the 19Ag gene demonstrated that it plays no essential role in growth and pathogenicity of M. intracellulare; however, the exact nature of its biological function remains unknown.

Identification of the Mycobacterium tuberculosis GlnE promoter and its response to nitrogen availability

Microbiology ◽  
2006 ◽  
Vol 152 (9) ◽  
pp. 2727-2734 ◽  
Author(s):  
Carey A. Pashley ◽  
Amanda C. Brown ◽  
Dina Robertson ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Nitrogen Availability ◽  
Essential Gene ◽  
Transcriptional Start Site ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Ammonia Assimilation ◽  
Directed Mutagenesis ◽  
Key Enzyme

Adenylyltransferase, GlnE, has a predicted role in controlling the enzymic activity of glutamine synthetase, the key enzyme in ammonia assimilation. It was previously demonstrated that glnE is an essential gene in Mycobacterium tuberculosis. glnE is located downstream of glnA2, one of four glutamine synthetases. The expression of GlnE under various conditions was determined. Although a co-transcript of glnA2 and glnE was detectable, the major transcript was monocistronic. A transcriptional start site immediately upstream of glnE was identified and it was shown by site-directed mutagenesis that the predicted −10 region is a functional promoter. It was demonstrated that in a Mycobacterium smegmatis background M. tuberculosis PglnE was up-regulated in ammonia- or glutamine-containing media.

scholarly journals Integrating bioinformatics tools to investigate protein phosphorylation

2015 ◽  
Author(s):  
Dimitrios Vlachakis ◽  
Elena Bencurova ◽  
Mangesh Bhide ◽  
Sophia Kossida
Keyword(s):  
Mass Spectrometry ◽  
Western Blot ◽  
Protein Phosphorylation ◽  
Gel Electrophoresis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Phosphorylation Sites ◽  
Post Translational Modifications ◽  
Cellular Processes ◽  
Benchmark Study

Protein phosphorylation is one of the most important protein post-translational modifications and plays a role in numerous cellular processes including recognition, signaling and degradation. It can be studied experimentally by various methodologies, like employing western blot analysis, site-directed mutagenesis, 2 D gel electrophoresis, mass spectrometry etc. A number of in silico tools have also been developed in order to predict plausible phosphorylation sites in a given protein. In this review, we conducted a benchmark study including the leading protein phosphorylation prediction software, in an effort to determine which performs best. The first place was taken by GPS 2.2, having predicted all phosphorylation sites with a 83% fidelity while in second place came NetPhos 2.0 with 69%.

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