Eukaryotic translation elongation factor 1A (eEF1A) domain I from S. cerevisiae is required but not sufficient for inter-species complementation

Ethanolamine phosphoglycerol (EPG) is a protein modification attached exclusively to eukaryotic elongation factor 1A (eEF1A). In mammals and plants, EPG is linked to conserved glutamate residues located in eEF1A domains II and III, whereas in the unicellular eukaryote Trypanosoma brucei, only domain III is modified by a single EPG. A biosynthetic precursor of EPG and structural requirements for EPG attachment to T. brucei eEF1A have been reported, but nothing is known about the EPG modifying enzyme(s). By expressing human eEF1A in T. brucei, we now show that EPG attachment to eEF1A is evolutionarily conserved between T. brucei and Homo sapiens. In contrast, S. cerevisiae eEF1A, which has been shown to lack EPG is not modified in T. brucei. Furthermore, we show that eEF1A cannot functionally complement across species when using T. brucei and S. cerevisiae as model organisms. However, functional complementation in yeast can be obtained using eEF1A chimera containing domains II or III from other species. In contrast, yeast domain I is strictly required for functional complementation in S. cerevisia

TORC2 downstream signaling in Saccharomyces cerevisiae

During their lives, eukaryotic cells need to adjust their metabolism and growth in response to changes of the surrounding conditions. These cellular responses are tightly coordinated and are required for cell survival and essential for the health of a multicellular organism. A major controller of growth as well as aging related processes is the serine/threonine TOR (Target Of Rapamycin) kinase. This highly conserved protein kinase is linked to many diseases and the model organism Saccharomyces cerevisiae has been a valuable tool in discovering and functionally characterizing this kinase. Unlike most other eukaryotes, S. cerevisiae encodes two TOR genes. TOR1 encodes the Tor1 kinase found in a multiprotein complex called TORC1-A only (Target Of Rapamycin Complex 1A), whereas the TOR2 gene encodes the Tor2 kinase found in TORC1-B and in TORC2. While TORC1-A and TORC1-B are inhibited by the macrocyclic lactone rapamycin, a TORC2 inhibitor has been missing, as well as the reason for its resistance to rapamycin. Consequently, the functions performed by TORC1 (including TORC1-A and TORC1-B) have been well characterized, whereas our understanding of TORC2 signaling remained preliminary. However, research of the past few years has revealed that both TOR complexes regulate their readouts through feedback mechanisms. In this thesis and in our JBC publication (Rispal et al., 2015), we present a tool in which TORC2 can be efficiently inhibited, leading to a model in which this complex regulates its downstream outputs actin cytoskeleton polarization and endocytosis via three pathways, including i) a fast and direct pathway where Ypk1 – a downstream target of TORC2 – could act as direct regulator of proteins involved in these processes; ii) further, an indirect regulator via the flippase kinases Fpk1 and Fpk2 might play a central role in coupling actin polarization and the endocytic machinery to TORC2. Lastly, iii) changes in the composition and especially depletion of complex sphingolipids from the plasma membrane indirectly signals to proteins mediating actin polarization and endocytosis. We also confirm the link of TORC2 to cell cycle progression and calcineurin signalling. Additionally, preliminary data indicates that actin organization and G2/M cell cycle progression could be mediated downstream of calcineurin. Moreover, it seems that TORC2 also influences inositol metabolism, a TORC2 function that hasn't been described yet. This highlights that TORC2 – like TORC1 – also exerts various functions within a yeast cell. We further wished to determine how TORC1 signals to TORC2 during inhibition with rapamycin. We observed that inhibition of TORC1-A is sufficient to induce TORC2 hyperactivation, as monitored by phosphorylation of Ypk1. However, the factors involved in this signalling event, as well as the physiological significance remain to be determined

TOR Complexes and the Maintenance of Cellular Homeostasis

The Target of Rapamycin (TOR) is a conserved serine/threonine (ser/thr) kinase that functions in two, distinct, multiprotein complexes called TORC1 and TORC2. Each complex regulates different aspects of eukaryote growth: TORC1 regulates cell volume and/or mass by influencing protein synthesis and turnover, while TORC2, as detailed in this review, regulates cell surface area by influencing lipid production and intracellular turgor. TOR complexes function in feedback loops, implying that downstream effectors are also likely to be involved in upstream regulation. In this regard, the notion that TORCs function primarily as mediators of cellular and organismal homeostasis is fundamentally different from the current, predominate view of TOR as a direct transducer of extracellular biotic and abiotic signals

Translation Elongation and Termination: 2 Are They Conserved Processes?

Translation initiation is followed by a process in which sequential addition of amino acid residues enables peptide chain formation, called translation elongation. Elongation decodes the codons on an mRNA and depends on elongation factors (EFs). In a first step, a ternary complex consisting of EF1A/EF-Tu-GTP-aminoacyl-tRNA (aa-tRNA) is formed and the elongator aa-tRNA is recruited to the ribosomal acceptor (A-) site. Hydrolysis of EF1A/EF-Tu-GTP is activated upon codon-anticodon decoding at the A-site and mRNA-tRNA interaction with the ribosome. Subsequently, peptide bond formation occurs between the aa moiety of the A-site aa-tRNA and the peptidyl-tRNA located at the ribosomal peptidyl (P-) site. Thereafter, the deacylated tRNA is moved to the exit (E-) site [1–4]. This elongating process is repeated until a stop codon (UAA, UAG and UGA) is encountered. Exposing a stop codon at the A-site initiates the process of Translation termination. Polypeptide release factors mediate the release of the polypeptide chain from the ribosome by hydrolysis of the ester bond between the polypeptide chain and the tRNA at the P-site [1–3, 5]

Complementation of chimeric eEF1A in <i>S. Cerevisiae.</i>

<p>(<b>A</b>) Sequence alignment of conserved amino acid motifs separating domains I and II (upper panel) and II and III (lower panel) of eEF1A from different sources. To generate chimeric constructs, synthetic SpeI (triangle) or BamHI (arrow) cloning sites were introduced. (<b>B</b>) Schematic representation of chimeric constructs. Arrows and triangles indicate the positions of the cloning sites which were removed by site-directed mutagenesis to reconstruct the original eEF1A sequences. Numbers in brackets correspond to clones shown in Fig. 3C. (<b>C</b>) Complementation of <i>S. cerevisiae</i> strain TKY102 with chimeric eEF1A constructs. Upper panels: Yeast cells growing on plates after transformation with different chimeric constructs. Middle and bottom panels: Counterselection for the loss of endogenous eEF1A on plates containing 5-FOA at 25 or 30°C. Left panels: Complementation assays with chimeric constructs carrying cloning sites causing a N329K mutation in the case of the artificial BamHI-site (separating domains II and III) or I254T/G255S mutations in the case of the artificial SpeI-site (separating domains I and II). Right panels: complementation assays with chimeric constructs after reconstructing wild type eEF1A sequence motifs. (<b>D</b>) Growth properties of <i>S. cerevisiae</i> complemented with chimeric eEF1A constructs. (1) positive control with yeast eEF1A; (2) Chimeric yeast constructs carrying human domain III - without or with N329K mutation; (4) Chimeric yeast constructs carrying <i>T. brucei</i> domain III - without or with N329K mutation; (8) Chimeric yeast eEF1A construct carrying humain domain II.</p

Characteristic ions of the tryptic fragments of eEF1A proteins detected by mass spectrometry.

<p>HA-eEF1A proteins expressed in <i>T. brucei</i> were purified, digested with trypsin and subjected to nano-LC-MS/MS as described in Materials and Methods. Purified carboxy-terminally His_6x-tagged <i>S. cerevisiae</i> eEF1A was treated prior to nano-LC-MS/MS the same way as for HA-tagged eEF1A proteins. Tryptic fragments containing the site of potential EPG attachment E362, E298/E372, E301/E374 of domainII/domain III from <i>T. brucei</i>, <i>S. cerevisiae</i> and <i>H. sapiens</i> eEF1A, respectively (all marked with an asterisk) are shown with their corresponding [M+H]⁺, [M+H]²⁺ and [M+H]³⁺ ions. The last column indicates the presence (+) or absence (−) of EPG modifications based on ion data.</p><p>n.d., not detected.</p><p>−,not present.</p><p>Ox, oxidation.</p>a<p>,described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042338#pone.0042338-Greganova2" target="_blank">[15]</a>.</p>b<p>,expressed as HA-tagged protein in <i>T. brucei.</i></p>c<p>,expressed as His_6x-tagged protein in <i>S. cerevisiae.</i></p>d<p>,the relative intensities of the [M+H]⁺ ions of the EPG-modified (<i>m/z</i> 1020.465) and unmodified (<i>m/z</i> 823.420) tryptic peptides suggest that >95% of <i>T. brucei</i> eEF1A is modified with EPG (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042338#pone.0042338-Greganova2" target="_blank">[15]</a>).</p

Expression and [³H]Etn-labeling of eEF1A orthologs in <i>T. Brucei.</i>

<p>(<b>A</b>) The predicted three-dimensional structures of eEF1A from <i>T. brucei</i> (left), <i>H. sapiens</i> (middle) and the X-ray structure of <i>S. cerevisiae</i> (right) are illustrated to document structural similarities. The positions of the glutamate residues representing potential EPG modification sites are indicated. The nomenclature of domains I, II, III is indicated for <i>S. cerevisiae</i> eEF1A. (<b>B</b>) <i>T. brucei</i> Δprocyclin#1 expressing HA-tagged human (HA-HsEF1A) or yeast (HA-ScEF1A) eEF1A were incubated in the presence of [³H]Etn for 18 h. Proteins in cell lysates (L) and in supernatants (SN), wash solutions (W) and the final pellet after immunoprecipitation using anti-HA antibody (IP) were separated by SDS-PAGE and analyzed by immunoblotting using α-HA monoclonal antibody (α-HA; upper panels) or fluorography (lower panels). Lanes contain extracts from 1×10⁷ (for L, SN and W) or 1.8×10⁸ cell equivalents (for IP). Molecular mass markers (kDA) are indicated.</p

<i>In vivo</i> complementation assays in <i>T. brucei</i> and <i>S. cerevisiae</i> depleted for endogenous eEF1A.

<p>(<b>A</b>) <i>T. brucei</i> RNAi parasites expressing ectopic copies of TbEF1A, HsEF1A, ScEF1A or LmEF1A were cultivated in the absence (−) or presence (+) of tetracycline (tet) for 7 days. Each day, cultures were diluted to a cell density of 3×10⁶ cells/ml and incubated with fresh medium. Non-induced HsEF1A, ScEF1A and LmEF1A cell lines showed the same growth curve as non-induced cell line TbEF1A: for simplicity, only the growth curve for TbEF1A is shown (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042338#pone.0042338-Greganova3" target="_blank">[20]</a>). (<b>B</b>) Northern blots of total RNA extracted from parasites after 3 days of incubation in the absence (−) or presence (+) of tetracycline (tet) and hybridized with ³²P-labeled probes against the intergenic region 1 of <i>T. brucei</i> eEF1A (top); rRNA was used as a loading control (bottom). (<b>C</b>) RT-PCR analysis of eEF1A transcripts. cDNA was synthesized from transcripts of <i>T. brucei</i> RNAi parasites cultured in the absence (−) or presence (+) of tetracycline for 72 h using primers specific for the different eEF1A orthologs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042338#pone.0042338.s002" target="_blank">Table S1b</a>). Lanes containing cDNA or total RNA (negative controls) are indicated. (<b>D</b>) Complementation assays in <i>S. cerevisiae</i> strain TKY102 expressing as unique source endogenous eEF1A from a URA3-plasmid. Cells were transformed with plasmids carrying genes encoding for different eEF1A orthologs. Upon transformation (upper panel), cells were incubated for several days on a plate containing 5-fluoroorotic acid (5-FOA) which is toxic in the presence of the URA3 plasmid. Only transformants which were able to loose due to mitotic segregation the URA3-plasmid grew on 5-FOA containing medium (lower panel). The numbers represent wild-type ScEF1A (1), HA-TbEF1A (2), TbEF1A (3), LmEF1A (4), HsEF1A (5), vector pRS314 (6, negative control), CaEF1A (7), His_6x-ScEF1A (8), and ScEF1A-His_6x (9).</p

Target of Rapamycin Complex 2 Regulates Actin Polarization and Endocytosis via Multiple Pathways

Target of rapamycin is a Ser/Thr kinase that operates in two conserved multiprotein complexes, TORC1 and TORC2. Unlike TORC1, TORC2 is insensitive to rapamycin, and its functional characterization is less advanced. Previous genetic studies demonstrated that TORC2 depletion leads to loss of actin polarization and loss of endocytosis. To determine how TORC2 regulates these readouts, we engineered a yeast strain in which TORC2 can be specifically and acutely inhibited by the imidazoquinoline NVP-BHS345. Kinetic analyses following inhibition of TORC2, supported with quantitative phosphoproteomics, revealed that TORC2 regulates these readouts via distinct pathways as follows: rapidly through direct protein phosphorylation cascades and slowly through indirect changes in the tensile properties of the plasma membrane. The rapid signaling events are mediated in large part through the phospholipid flippase kinases Fpk1 and Fpk2, whereas the slow signaling pathway involves increased plasma membrane tension resulting from a gradual depletion of sphingolipids. Additional hits in our phosphoproteomic screens highlight the intricate control TORC2 exerts over diverse aspects of eukaryote cell physiology

Molecular Basis of the Rapamycin Insensitivity of Target Of Rapamycin Complex 2

Target of Rapamycin (TOR) plays central roles in the regulation of eukaryote growth as the hub of two essential multiprotein complexes: TORC1, which is rapamycin-sensitive, and the lesser characterized TORC2, which is not. TORC2 is a key regulator of lipid biosynthesis and Akt-mediated survival signaling. In spite of its importance, its structure and the molecular basis of its rapamycin insensitivity are unknown. Using crosslinking-mass spectrometry and electron microscopy, we determined the architecture of TORC2. TORC2 displays a rhomboid shape with pseudo-2-fold symmetry and a prominent central cavity. Our data indicate that the C-terminal part of Avo3, a subunit unique to TORC2, is close to the FKBP12-rapamycin-binding domain of Tor2. Removal of this sequence generated a FKBP12-rapamycin-sensitive TORC2 variant, which provides a powerful tool for deciphering TORC2 function in vivo. Using this variant, we demonstrate a role for TORC2 in G2/M cell-cycle progression