Figure 1.
Amino acid sequence comparison around the EPG modification site.
The amino acid sequence of T. brucei eEF1A between residues 347 and 373 is compared to the corresponding eEF1A sequences from Daucus carota (GenBank, BAA02205) and Mus musculus (GenBank, NP_034236) using ClustalW. A grey background indicates amino acid identity between the sequences. The arrows in indicate the conserved residues at, or around, the EPG modification site of T. brucei eEF1A (Glu362) that were mutated in this study (E362Q, E362A, E362D, F360T, A361D, K366S). The box marks the tryptic fragments containing the EPG modification site (indicated by the asterisk).
Figure 2.
Expression of mutated HA-eEF1A and labeling with [3H]Etn.
T. brucei Δprocyclin#1 expressing HA-eEF1A carrying the point mutations E362Q, E362A or E362D were incubated in the presence of [3H]Etn for 18 h. HA-eEF1A in the cell lysates (L) and after immunoprecipitation (IP) were analyzed by SDS-PAGE using 12% acrylamide gels, followed by immunoblotting using α-HA antibody (upper panels), or fluorography (lower panels). The lanes contain material from 1×107 parasites, except for the lanes with the immunoprecipitates in the fluorographs, which contain material prepared from 1.8×108 cell equivalents.
Figure 3.
Replacement of a T. brucei eEF1A peptide sequence by its yeast homologue.
(A) The primary sequence of T. brucei eEF1A between residues 347 and 373 is aligned to the corresponding eEF1A sequence from S. cerevisiae (GeneDB, YBR118W) using ClustalW (http://clustalw.genome.ad.jp). A grey background indicates amino acid identity between the sequences. The arrows indicate the residues that were sequentially mutated in T. brucei eEF1A (A361D, I363L, E364L, S365E) to substitute the stretch between Phe360 and K366 (FAEIESK) by the corresponding S. cerevisiae sequence (FDELLEK). The asterisk indicates the EPG modification site and the frame marks the tryptic fragments analyzed by mass spectrometry. (B) T. brucei Δprocyclin#1 expressing HA-eEF1A, containing the yeast sequence FDELLEK instead of FAEIESK, were incubated with [3H]Etn for 18 h. Proteins in the cell lysate (L), supernatant (SN), wash solution (W) and immunoprecipitate (IP) were separated and analyzed by immunoblotting as described in Fig. 2. The lanes contain material from 1×107 parasites, except for the lanes with the immunoprecipitates in the fluorographs, which contain material prepared from 1.8×108 cell equivalents.
Figure 4.
Analysis of EPG attachment by mass spectrometry.
Tryptic peptides from T. brucei HA-eEF1A mutants were subjected to nano-LC-MS/MS and the collision-induced fragmentation spectra of ions m/z 510.736 and 405.206, corresponding to EPG-modified FAE*IESK and unmodified FADIESK, respectively, are shown. The intensity scale in panel A is set to 12.5% of the most abundant ion of 424.718, which corresponds to the doubly charged parent ion after neutral loss of glycerol phosphate. The y and b type ions are marked with the corresponding m/z of the singly charged ions given above or underneath the amino acid sequence.
Table 1.
Characteristic ions of the tryptic fragments of T. brucei eEF1A proteins detected by mass spectrometry.
Figure 5.
Tertiary structure of T. brucei eEF1A and design of deletion mutants.
(A) Predicted three-dimensional structure of T. brucei eEF1A using PyMol (http://www.pymol.org). The site of EPG attachment, Glu362, is exposed on the surface of domain III. Domain III is highlighted in colour; the amino acid sequence common to the minimal structural units showing EPG attachment (residues 348–394) is indicated in red, the flanking N- and C-terminal stretches (residues 315–347 and 395–449, respectively) are in yellow. (B) Overview of HA-tagged deletion mutants of T. brucei eEF1A. The numbers indicate the positions of the amino acids in the full length protein. The black boxes represent the HA-tags and the asterisks indicate the EPG modification site (Glu 362). Predicted molecular masses are indicated in kDa on the right.
Figure 6.
Expression of HA-eEF1A deletion mutants and labeling with [3H]Etn.
T. brucei Δprocyclin#1 expressing full length HA-eEF1A, or HA-tagged deletion mutants, were incubated in the presence of [3H]Etn for 18 h. Subsequently, parasites were lysed, and HA-tagged proteins were immunoprecipitated and analyzed by glycine-SDS-PAGE (left panels) or Tricine-SDS-PAGE (right panels). HA-eEF1A was visualized by immunoblotting using α-HA (upper panels), whereas labeling with [3H]Etn was analyzed by fluorography (lower panels). The lanes contain material from 1×107 parasites, except for the lanes with the immunoprecipitates in the fluorographs, which contain material prepared from 1.8×108 cell equivalents. The deletion mutants are marked according to Fig. 5B. Molecular mass markers are indicated in kDa.
Figure 7.
Expression of domain III fusion mutants and labeling with [3H]Etn.
(A), (B), T. brucei Δprocyclin#1 expressing HA-Alba-III and PTP-III fusion proteins were labeled with [3H]Etn for 18 h. Proteins in the cell lysates (L), supernatants (SN), wash solutions (W) and immunoprecipitates (IP) were analyzed by SDS-PAGE using 12% acrylamide gels. SDS PAGE was followed either by immunoblotting (upper panels) or fluorography (lower panels). For immunoblotting α-HA antibody or α-IgG were used to detect HA-Alba-III or PTP-III, respectively. The lanes contain material from 1×107 parasites, except for the lanes with the immunoprecipitates in the fluorographs, which contain material prepared from 1.8×108 cell equivalents.