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. 2016 Apr 11;60(2):2612.
doi: 10.4081/ejh.2016.2612.

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  1 , P Kwaśniak, K Miller, K Kobyłecki, M Osińska
Affiliations

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 et al. Eur J Histochem. .

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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Conflict of interest statement

Conflict of interest: the authors report no conflics of interest.

Figures

Figure 1.
Figure 1.
Recombinant Rab7b variants undergo glycosylation in the presence of Paramecium octaurelia cell homogenate and UDP-glucose or [C]UDP-glucose. A) Recombinant (His)6Rab7b proteins were exposed to UDP-glucose and cell homogenate as described in Materials and Methods; after electrophoretic separation of the eluted proteins and Ponceau S staining, Con A overlay assay was performed followed by stripping and subsequent Western blotting with anti-His antibody and - after next stripping – with specific anti-Rab7b antipeptide antibody; molecular mass marker (kDa) is shown at the left. B) In vitro assay with [C]UDP-glucose, recombinant variants and Paramecium homogenate; incorporation into (His)6Rab7b_200 as compared with the control (His)6Rab7b; densitometry of bands; intensities of the (His)6Rab7b_200 protein bands of the Coomassie Blue stained SDS PAGE were calculated as relative optical density vs. (His)6Rab7b as the control sample. Asterisk indicates significant difference from (His)6Rab7b at P<0.1, n=4.
Figure 2.
Figure 2.
Comparison between averaged spectra from the nanoLC-MS separation of glycosylated (His)6Rab7b after UDP-glucose incorporation as described in Materials and Methods (A) with the control recombinant (His)6 Rab7b incubated without UDP-glucose under the same experimental conditions (B). There is the peptide ion at a mass to charge (m/z) ratio of = 677.6 in the spectrum (A, red arrow) not detected after deglycosylation (B). Inset in (A) shows a blow-up of this peptide ion. Isotopic pattern allows for estimation of ionization ratio as 3+. Both averaged spectra obtained by the accumulation of ca. 40 scans acquired within retention time: 21.4-22.2 min.
Figure 3.
Figure 3.
MS/MS spectrum derived after fragmentation of the 677.63+ ion detected in glycosylated Rab7b shown in Figure 2. Blue ions: come directly from the spectrum, red ions are derived after deconvolution of the blue ions; green ions: most likely represent hybrids of glycan modification and peptide fragments. In the upper part of the spectrum b-ions and y-ions are marked with corresponding amino acid residues. Additionally, identified sequence of the [195-206] peptide is shown: identification of the exact amino acid residues was based on y-ions (blue residue), b-ions (red residues) or both series (green residues). Black font indicates the single unidentified residue.
Figure 4.
Figure 4.
Incorporation of [P] into recombinant (His)6Rab7b and its mutagenized form (His)6Rab7b_200 in the presence of Paramecium octaurelia cell homogenate. A) Upper panel, Coomassie stained gel (MW marker at the left); lower panel, autoradiogram. B) Equal volumes of eluted recombinant proteins were immediately analyzed in a scintillation counter and their identical aliquots subjected to SDS PAGE; densitometry of bands; intensities of the (His)6Rab7b_200 protein bands were calculated as relative optical density vs (His)6Rab7b control samples. Asterisk represents a significant difference from the control sample at P<0.01 by Student’s t-test, n=3.
Figure 5.
Figure 5.
Recombinant (His6) Rab7b variants undergo in vitro phosphorylation. The same blot analysis using antibodies against phosphorylated amino acids. The experiment was conducted as described in Figure 1A. After electrophoretic separation of eluted affinity purified proteins by 15% SDS PAGE and Ponceau Red staining, Western blotting was performed sequentially with anti P-Tyr, anti P-Ser, anti His-tag and anti-Rab7b specific antipeptide Ab. Stripping at each step was performed as described in Materials and Methods. Molecular mass marker is shown at the left.
Figure 6.
Figure 6.
Phosphorylation patterns of Paramecium octaurelia Rab7a and Rab7b proteins detected by nano LC-MS/MS.
Figure 7.
Figure 7.
In vivo incorporation of recombinant proteins (His)6Rab7b and (His)6Rab7b_200 into electroporated Paramecium octaurelia cells. Immunolocalization in a STED confocal microscope. Recombinant proteins detected with anti-His antibody conjugated with rhodamine (red), detection of tubulin with antibody conjugated with FITC (green). A) (His)6Rab7b, incorporation of the recombinant protein within the cytostome area (arrow). B) (His)6Rab7b_200; please note dispersed incorporation of the protein; scale bars: 10 µm. C,D,E) (His)6Rab7b. C,D) Two consecutive confocal optical slices (1 µm) showing accumulation of (His)6Rab7b at the edge of the cytostome (white arrowhead); scale bars: 10 µm. E) Tubulin association with the incorporated control recombinant (His)6Rab7b protein within the cytostome is visible at higher magnification (white arrow); scale bars: 5 µm.
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Grants and funding

Funding: this work was supported by statutory funds to the Nencki Institute of Experimental Biology of Polish Academy of Sciences and by grant N. N303 615038 from the Ministry of Science and Higher Education/National Science Centre.