Figure 1.
Multiple Sequence alignment of 14-3-3 proteins.
Homo sapiens 14-3-3epsilon (h14-3-3ε, accession number P62258.) and 14-3-3zeta (h14-3-3ζ, P29312); Drosophila melanogaster D14-3-3epsilon (D14-3-3ε, P92177) and Leonardo II (LeoII, P29310-2); Giardia duodenalis 14-3-3 (g14-3-3, AAZ91664.1). Invariant or conserved amino-acids are boxed in black, those conserved in at least two proteins are highlighted in gray and divergent ones are left unboxed. Dashes indicate gaps. Stars indicate residues that in the human 14-3-3zeta form salt bridges involved in N-terminal dimerization (Arg18-Glu89, Glu5-Lys74, and Asp21-Lys85).
Figure 2.
Cross species expression and subcellular localization of 14-3-3 proteins.
A) Expression of Drosophila 14-3-3s in Giardia. Approximately 7 µg of proteins extracted from transfected trophozoites (T, or Troph.) and 12 hr encysting parasites (Encyst.) were separated on 12,5% SDS-PAGE, transferred onto a PVDF membrane and probed with anti-FLAG mAb. Untransfected WB-C6 and the g14-3-3 transgenic line were used as controls. B) Expression of g14-3-3 in Drosophila. Two adult flies per genotype were homogenized in 50µl Laemmli buffer and 10µl homogenate per lane was resolved on 12% SDS-PAGE and transferred onto a PVDF membrane and probed with the indicated antibodies. The neuronal protein Syntaxin (syx) was used to ascertain equal loading. The w1118 strain, parental to the tranformants was used as control. C) Subcellular localization of the FLAG-tagged Drosophila 14-3-3s compared to g14-3-3-FLAG in Giardia trophozoites and 12h encysting parasites encysting parasite. Parasites were stained with Cy3-conjugated anti-FLAG mAb (red), rabbit anti-g14-3-3 serum (N14) followed by Alexa Fluor-488 anti-rabbit (green) or with FITC-conjugated anti-CWP mAb (green), and with DAPI (blue). Transmission light acquisition (T). Scale bars, 2.5 µm. D) g14-3-3 expression in Drosophila embryonic and adult neurons as indicated. The neuronal-specific nuclear protein Elav is red (rat anti-Elav revealed by anti-rat Alexa Fluor-555), while g14-3-3-His is green (mouse anti-His revealed by anti-mouse Alexa Fluor-488). The arrow points to an embryonic motor neuron axon which contains only the cytoplasmic His-g14-3-3. The far right panel is a magnification of the middle panel revealing g14-3-3 expression in adult mushroom body neurons.
Figure 3.
Selective heterodimerization of Giardia and Drosophila 14-3-3s.
A) Coomassie stained 12% SDS-PAGE of affinity purified FLAG-tagged 14-3-3s from transfected Giardia and WB-C6 controls. The expected position of the endogenous g14-3-3 and FLAG-tagged proteins is indicated. The lower band visible in the FLAG-D14-3-3ε was identified as D14-3-3ε, representing a proteolytic product likely at N-terminus. B) Western blot analysis using 1∶10 of the affinity purified FLAG-tagged 14-3-3s separated on a 12% SDS-PAGE. The membrane was sequentially probed with anti-FLAG mAb (left panel) and then with the N14 anti-g14-3-3 serum. Untransfected WB-C6 were used as controls. C) Western blot analysis using 1∶10 of the affinity purified His-tagged g14-3-3 separated on a 12% SDS-PAGE. The transgenic protein was expressed either throughout the adult fly (TubG4) or specifically in the CNS (ElavG4). For the CNS expressed protein adult head lysates were used exclusively. The expected locations of the endogenous Drosophila proteins are indicated. Both blots were probed simultaneously with the anti-Leo and anti-D14-3-3ε antibodies. Driver alone heterozygotes (TubG4>+ or ElavG4>+) were used as controls. D) Whole fly lysates from the indicated control and His-g14-3-3 expressing animals were cross-linked with BS3. Complexes were separated on a 10% SDS-PAGE without (left two panels) or after His-tag affinity purification through Ni beads and the blots probed with the indicated antibodies. The membranes were probed with the indicated antibodies. The expected electrophoretic mobilities of monomers and dimers are indicated.
Figure 4.
Phosophorylation and polyglycylation of 14-3-3 proteins in Giardia.
A) Multiple Alignments of g14-3-3, LeoII and D14-3-3ε amino-acid sequences showing the peptides derived from trypsin digestion and containing the phosphorylated Thr214 (black boxed) and the Glu246 (grey boxed) of g14-3-3 and the corresponding peptides of LeoII and D14-3-3ε are in bold. Residues are numbered according to published protein sequences. B) Alignment of the C-terminus of g14-3-3, LeoII and D14-3-3ε with α- and β-tubulin of Giardia (GenBankTM/EBI Accession Number AAN46106 and P05304), α- and β-tubulin of Paramecium tetraurelia (GenBankTM/EBI Accession Number CAA67848 and CAE75646), and α- and β-tubulin of Tetrahymena thermophila (GenBankTM/EBI Accession Number P41351 and P41352). The alignment was performed with the ClustalW program and manually refined. The amino acids in grey define the hypothetical polyglycylation sequence [T/G]X0-1[D/E]X1-3G[D/E]X1-2[E]2-4. Experimentally defined polyglycylated glutamic acid residues are black boxed and highlighted in bold white letters. Putative polyglycylated glutamic acid residues are only black boxed. The underlined glutamic acid of Tetrahymena α-tubulin is predicted to be polyglycylated. C-E) MALDI-MS analysis of affinity purified FLAG-tagged transfected proteins from Giardia trophozoites. MALDI-MS spectra of FLAG-g14-3-3 (C), FLAG-D14-3-3ε (D) and FLAG-LEOII (E), encompassing the MH+ range of 1400–3600. Mono-isotopic masses of relevant peaks are shown. Peptides are indicated by the positions of their N- and C-termini and numbered as in the protein sequence. Peaks shifted from the theoretical MH+ are indicated by arrows as for the 80 Da shift due to phosphorylation. For each protein, analysis of phosphorylation is reported on the left and the analysis of C-terminal polyglycylation is reported on the right panels. C) For FLAG-g14-3-3, the calculated MH+ peak for peptide 202–219 at 2029.9 is clearly shifted to 2109.93 indicating phosphorylation on Thr214 (202AFDAAITDLDKLTEESYK219). On the right, the peaks corresponding to polyglycylation of the peptide (230DNLNLWVTDSAGDDNAEEK248) with predicted MH+ = 2047.92 was clearly shifted to 2105.9 indicating multiple glycines on Glu246 as revealed by their number in the lateral chain. The insert shows the lack of the peak corresponding to the unmodified 230-248 peptide. D) For D14-3-3ε-FLAG, the predicted peak at MH+ = 2087.9 for the peptide (197AAFDDAIAELDTLSEESYK21) was shifted by 80 kDa to MH+ = 2167.9, indicating phosphorylation at Ser210. On the right, the peaks corresponding to the unmodified (249EQIQDVEDQDVS260 = 1404.6 MH+) peptide and the shifts to higher MH+ consistent with addition of 7 or 8 glycines (right) and phosphorylation at Ser260 (+80kDa, arrow on the left) are shown. C) For LEOII-FLAG only the peaks corresponding to unmodified (197QAFDDAIAELDTLNEDSYK215 = 2157.98 MH+) and (226DNLTLWTSDTQGDEAEPQEGGDN248 = 2492.03 MH+) peptides were evident.
Figure 5.
MALDI-MS analysis of affinity purified endogenous and transgenic g14-3-3 proteins from Drosophila.
A) Western blot analyses of GST-difopein purified 14-3-3s (1∶10) from wild type flies and His-g14-3-3 -expressing transgenic flies and the relevant controls (WB-C6 for Giardia and w1118 for flies) were separated on 12% SDS-PAGE, blotted and the membranes probed with the indicated antibodies. The AXO49 mAb detecting the Giardia polyglycylated protein did not cross react with the fly isoforms, which are detected with the anti-pan14-3-3 antibody, while the transgenic g14-3-3 protein is detectable with the anti-His in fly lysates. TR denotes trophozoite lysates, while ENC indicates lysates from 12hr encysting parasites. MALDI-MS spectra of the transgenic His-g14-3-3 (B) and the endogenous Drosophila D14-3-3ε (C) and Leo (D) encompassing the MH+ range of 1400-3600. Mono-isotopic masses of relevant peaks are shown. Peptides are indicated by the positions of their NH2- and C-termini and numbered as in the protein sequence. For each protein, the analysis of phosphorylation is reported on the left panels and the analysis of C-terminal polyglycylation is reported on the right panels. Only peaks corresponding to the unmodified peptides were visible and reported here.
Figure 6.
Functional consequences of exogenous 14-3-3 expression in Giardia and Drosophila.
A) The number of trophozoites, encysting parasites and cysts expressing the FLAG-tagged Drosophila 14-3-3s were estimated by co-staining with anti-CWP and anti-FLAG antibodies after 12 h of growth in encysting medium and counting. Their numbers were compared to those of similarly cultured parasites expressing g14-3-3-FLAG. Results from three independent experiments are reported and the bars represent the total number of cells observed (approximately 1000 parasites per experiment/transfected line) and the error is represented as the standard deviation. B) The number of adult flies homozygous for the null mutation D14-3-3εex4 alone or expressing g14-3-3 ubiquitously is reported relative to controls arbitrarily set to 100. Error bars are standard errors of the mean. C) Semi-quantitative Western blots from late embryonic extracts of the levels of endogenous Drosophila 14-3-3s in homozygotes for the D14-3-3εex4 mutation alone or expressing g14-3-3 ubiquitously. Both anti-Leo and anti-D14-3-3ε antibodies were applied simultaneously. The level of Tubulin in the lysates was used to control for loading. The ratio of each endogenous 14-3-3 to Tub in control animals was arbitrarily set to 1 and similarly calculated ratios in the experimental genotypes are reported relative to it. The blot is representative of three in total and error bars in the graphs represent the standard errors of the mean.