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

  • E. Wyroba | e.wyroba@nencki.gov.pl Nencki Institute of Experimental Biology of Polish Academy of Sciences, Poland.
  • P. Kwaśniak Nencki Institute of Experimental Biology of Polish Academy of Sciences, Poland.
  • K. Miller Nencki Institute of Experimental Biology of Polish Academy of Sciences, Poland.
  • K. Kobyłecki Nencki Institute of Experimental Biology of Polish Academy of Sciences, Poland.
  • M. Osińska Nencki Institute of Experimental Biology of Polish Academy of Sciences, Poland.

Abstract

Protein products of the paralogous genes resulting from the whole genome duplication may acquire new function. 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 Ala200 resulted in diminished incorporation of [P32] by 37.4% and of 32 [C14] UDP-glucose by 24%, respectively, 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 contrary to non-mutagenized recombinant Rab7b correctly incorporated in the cytostome area. We identified the peptide ion at m/z=677.63+ characteristic for the glycan group attached to Thr200 in Rab7b using nano LC-MS/MS and comparing the peptide map of this protein with that after deglycosylation with the mixture of five enzymes of different specificity. Based on the mass of this peptide ion and quantitative radioactive assays with [P32]and  [C14] UDP-glucose, the suggested composition of the adduct attached to Thr200 might be (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 Rab7b in Paramecium is crucial for the proper localization/function of this protein. Moreover, these proteins differ also in other PTM: the number of phosphorylated amino acids in Rab7b is much higher than in Rab7a.

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Author Biography

E. Wyroba, Nencki Institute of Experimental Biology of Polish Academy of Sciences

Department Cell Biology,

Head, Laboratory of Cell Membrane Physiology

 

Published
2016-04-11
Section
Original Papers
Supporting Agencies
Nencki Institute of Experimental Biology of Polish Academy of Sciences, Ministry of Science and Higher Education/National Science Centre Grant N. N303 615038
Keywords:
Point mutagenesis, electroporation, post-translational modifications, mass spectrometry, glycosylation, Rab7, Paramecium, recombinant proteins.
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How to Cite
Wyroba, E., Kwaśniak, P., Miller, K., Kobyłecki, K., & Osińska, M. (2016). 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. European Journal of Histochemistry, 60(2). https://doi.org/10.4081/ejh.2016.2612