Cargando…

Site-Directed Mutagenesis, in Vivo Electroporation and Mass Spectrometry in Search for Determinants of the Subcellular Targeting of Rab7b Paralogue in the Model Eukaryote Paramecium Octaurelia

Protein products of paralogous genes resulting from whole genome duplication may acquire new functions. The role of post-translational modifications (PTM) in proper targeting of Paramecium Rab7b paralogue (distinct from that of Rab7a directly involved in phagocytosis) was studied using point mutagen...

Descripción completa

Detalles Bibliográficos
Autores principales: Wyroba, E., Kwaśniak, P., Miller, K., Kobyłecki, K., Osińska, M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: PAGEPress Publications, Pavia, Italy 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4933825/
https://www.ncbi.nlm.nih.gov/pubmed/27349314
http://dx.doi.org/10.4081/ejh.2016.2612
_version_ 1782441229954318336
author Wyroba, E.
Kwaśniak, P.
Miller, K.
Kobyłecki, K.
Osińska, M.
author_facet Wyroba, E.
Kwaśniak, P.
Miller, K.
Kobyłecki, K.
Osińska, M.
author_sort Wyroba, E.
collection PubMed
description Protein products of paralogous genes resulting from whole genome duplication may acquire new functions. The role of post-translational modifications (PTM) in proper targeting of Paramecium Rab7b paralogue (distinct from that of Rab7a directly involved in phagocytosis) was studied using point mutagenesis, proteomic analysis and double immunofluorescence after in vivo electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala diminished the incorporation of [P(32)] by 37% and of [C(14-)]UDP-glucose by 24% into recombinant Rab7b_200 in comparison to the non-mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells in contrast to non-mutagenized recombinant Rab7b correctly incorporated in the cytostome area. Using nano LC-MS/MS to compare the peptide map of Rab7b with that after deglycosylation with a mixture of five enzymes of different specificity we identified a peptide ion at m/z=677.6(3+) representing a glycan group attached to Thr200. Based on its mass and quantitative assays with [P(32)] and [C(14)]UDP-glucose, the suggested composition of the adduct attached to Thr200 is (Hex)1(HexNAc)1(Phos)3 or (HexNAc)1 (Deoxyhexose)1 (Phos)1 (HexA)1. These data indicate that PTM of Thr200 located in the hypervariable C-region of Paramecium octaurelia Rab7b is crucial for the proper localization/function of this protein. Moreover, the two Rab7 paralogues differ also in another PTM: substantially more phosphorylated amino acid residues are in Rab7b than in Rab7a.
format Online
Article
Text
id pubmed-4933825
institution National Center for Biotechnology Information
language English
publishDate 2016
publisher PAGEPress Publications, Pavia, Italy
record_format MEDLINE/PubMed
spelling pubmed-49338252016-07-15 Site-Directed Mutagenesis, in Vivo Electroporation and Mass Spectrometry in Search for Determinants of the Subcellular Targeting of Rab7b Paralogue in the Model Eukaryote Paramecium Octaurelia Wyroba, E. Kwaśniak, P. Miller, K. Kobyłecki, K. Osińska, M. Eur J Histochem Original Paper Protein products of paralogous genes resulting from whole genome duplication may acquire new functions. The role of post-translational modifications (PTM) in proper targeting of Paramecium Rab7b paralogue (distinct from that of Rab7a directly involved in phagocytosis) was studied using point mutagenesis, proteomic analysis and double immunofluorescence after in vivo electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala diminished the incorporation of [P(32)] by 37% and of [C(14-)]UDP-glucose by 24% into recombinant Rab7b_200 in comparison to the non-mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells in contrast to non-mutagenized recombinant Rab7b correctly incorporated in the cytostome area. Using nano LC-MS/MS to compare the peptide map of Rab7b with that after deglycosylation with a mixture of five enzymes of different specificity we identified a peptide ion at m/z=677.6(3+) representing a glycan group attached to Thr200. Based on its mass and quantitative assays with [P(32)] and [C(14)]UDP-glucose, the suggested composition of the adduct attached to Thr200 is (Hex)1(HexNAc)1(Phos)3 or (HexNAc)1 (Deoxyhexose)1 (Phos)1 (HexA)1. These data indicate that PTM of Thr200 located in the hypervariable C-region of Paramecium octaurelia Rab7b is crucial for the proper localization/function of this protein. Moreover, the two Rab7 paralogues differ also in another PTM: substantially more phosphorylated amino acid residues are in Rab7b than in Rab7a. PAGEPress Publications, Pavia, Italy 2016-04-11 /pmc/articles/PMC4933825/ /pubmed/27349314 http://dx.doi.org/10.4081/ejh.2016.2612 Text en ©Copyright E. Wyroba et al. http://creativecommons.org/licenses/by-nc/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Paper
Wyroba, E.
Kwaśniak, P.
Miller, K.
Kobyłecki, K.
Osińska, M.
Site-Directed Mutagenesis, in Vivo Electroporation and Mass Spectrometry in Search for Determinants of the Subcellular Targeting of Rab7b Paralogue in the Model Eukaryote Paramecium Octaurelia
title Site-Directed Mutagenesis, in Vivo Electroporation and Mass Spectrometry in Search for Determinants of the Subcellular Targeting of Rab7b Paralogue in the Model Eukaryote Paramecium Octaurelia
title_full Site-Directed Mutagenesis, in Vivo Electroporation and Mass Spectrometry in Search for Determinants of the Subcellular Targeting of Rab7b Paralogue in the Model Eukaryote Paramecium Octaurelia
title_fullStr Site-Directed Mutagenesis, in Vivo Electroporation and Mass Spectrometry in Search for Determinants of the Subcellular Targeting of Rab7b Paralogue in the Model Eukaryote Paramecium Octaurelia
title_full_unstemmed Site-Directed Mutagenesis, in Vivo Electroporation and Mass Spectrometry in Search for Determinants of the Subcellular Targeting of Rab7b Paralogue in the Model Eukaryote Paramecium Octaurelia
title_short Site-Directed Mutagenesis, in Vivo Electroporation and Mass Spectrometry in Search for Determinants of the Subcellular Targeting of Rab7b Paralogue in the Model Eukaryote Paramecium Octaurelia
title_sort site-directed mutagenesis, in vivo electroporation and mass spectrometry in search for determinants of the subcellular targeting of rab7b paralogue in the model eukaryote paramecium octaurelia
topic Original Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4933825/
https://www.ncbi.nlm.nih.gov/pubmed/27349314
http://dx.doi.org/10.4081/ejh.2016.2612
work_keys_str_mv AT wyrobae sitedirectedmutagenesisinvivoelectroporationandmassspectrometryinsearchfordeterminantsofthesubcellulartargetingofrab7bparalogueinthemodeleukaryoteparameciumoctaurelia
AT kwasniakp sitedirectedmutagenesisinvivoelectroporationandmassspectrometryinsearchfordeterminantsofthesubcellulartargetingofrab7bparalogueinthemodeleukaryoteparameciumoctaurelia
AT millerk sitedirectedmutagenesisinvivoelectroporationandmassspectrometryinsearchfordeterminantsofthesubcellulartargetingofrab7bparalogueinthemodeleukaryoteparameciumoctaurelia
AT kobyłeckik sitedirectedmutagenesisinvivoelectroporationandmassspectrometryinsearchfordeterminantsofthesubcellulartargetingofrab7bparalogueinthemodeleukaryoteparameciumoctaurelia
AT osinskam sitedirectedmutagenesisinvivoelectroporationandmassspectrometryinsearchfordeterminantsofthesubcellulartargetingofrab7bparalogueinthemodeleukaryoteparameciumoctaurelia