Vesicle transmembrane potential is required for translocation to the cytosol of externally added FGF-1

Externally added fibroblast growth factor-1 (FGF-1) is capable of crossing cellular membranes to reach the cytosol and the nucleus in a number of cell types. We have monitored the translocation of the growth factor by two methods: phosphorylation of FGF-1, and prenylation of an FGF-1 mutant that contains a C-terminal prenylation signal. Inhibition of endosomal acidification by ammonium chloride or monensin did not block the translocation of FGF-1, whereas bafilomycin A1, a specific inhibitor of vacuolar proton pumps, blocked translocation completely. A combination of ionophores expected to dissipate the vesicular membrane potential (valinomycin plus monensin) also fully inhibited the translocation. The inhibition of translocation by bafilomycin A1 was overcome in the presence of monensin or nigericin, while ouabain blocked translocation under these conditions. The data indicate that translocation of FGF-1 to cytosol occurs from the lumen of intracellular vesicles possessing vacuolar proton pumps, and that a vesicular membrane potential is required. Apparently, activation of vesicular Na(+)/K(+)-ATPase by monensin or nigericin generates a membrane potential that can support translocation when the proton pump is blocked

Characterization of the biochemical activity and tumor-promoting role of the dual protein methyltransferase METL-13/METTL13 in Caenorhabditis elegans

The methyltransferase-like protein 13 (METTL13) methylates the eukaryotic elongation factor 1 alpha (eEF1A) on two locations: the N-terminal amino group and lysine 55. The absence of this methylation leads to reduced protein synthesis and cell proliferation in human cancer cells. Previous studies showed that METTL13 is dispensable in non-transformed cells, making it potentially interesting for cancer therapy. However, METTL13 has not been examined yet in whole animals. Here, we used the nematode Caenorhabditis elegans as a simple model to assess the functions of METTL13. Using methyltransferase assays and mass spectrometry, we show that the C. elegans METTL13 (METL-13) methylates eEF1A (EEF-1A) in the same way as the human protein. Crucially, the cancer-promoting role of METL-13 is also conserved and depends on the methylation of EEF-1A, like in human cells. At the same time, METL-13 appears dispensable for animal growth, development, and stress responses. This makes C. elegans a convenient whole-animal model for studying METL13-dependent carcinogenesis without the complications of interfering with essential wild-type functions

Phosphorylation-regulated Nucleocytoplasmic Trafficking of Internalized Fibroblast Growth Factor-1

Fibroblast growth factor-1 (FGF-1), which stimulates cell growth, differentiation, and migration, is capable of crossing cellular membranes to reach the cytosol and the nucleus in cells containing specific FGF receptors. The cell entry process can be monitored by phosphorylation of the translocated FGF-1. We present evidence that phosphorylation of FGF-1 occurs in the nucleus by protein kinase C (PKC)δ. The phosphorylated FGF-1 is subsequently exported to the cytosol. A mutant growth factor where serine at the phosphorylation site is exchanged with glutamic acid, to mimic phosphorylated FGF-1, is constitutively transported to the cytosol, whereas a mutant containing alanine at this site remains in the nucleus. The export can be blocked by leptomycin B, indicating active and receptor-mediated nuclear export of FGF-1. Thapsigargin, but not leptomycin B, prevents the appearance of active PKCδ in the nucleus, and FGF-1 is in this case phosphorylated in the cytosol. Leptomycin B increases the amount of phosphorylated FGF-1 in the cells by preventing dephosphorylation of the growth factor, which seems to occur more rapidly in the cytoplasm than in the nucleus. The nucleocytoplasmic trafficking of the phosphorylated growth factor is likely to play a role in the activity of internalized FGF-1

The novel lysine specific methyltransferase METTL21B affects mRNA translation through inducible and dynamic methylation of Lys-165 in human eukaryotic elongation factor 1 alpha (eEF1A)

Lysine methylation is abundant on histone proteins, representing a dynamic regulator of chromatin state and gene activity, but is also frequent on many nonhistone proteins, including eukaryotic elongation factor 1 alpha (eEF1A). However, the functional significance of eEF1Amethylation remains obscure and it has remained unclear whether eEF1A methylation is dynamic and subject to active regulation. We here demonstrate, using a wide range of in vitro and in vivo approaches, that the previously uncharacterized human methyltransferase METTL21B specifically targets Lys-165 in eEF1A in an aminoacyl-tRNA- and GTP-dependent manner. Interestingly, METTL21B mediated eEF1A methylation showed strong variation across different tissues and cell lines, and was induced by altering growth conditions or by treatment with certain ER-stress-inducing drugs, concomitant with an increase in METTL21B gene expression. Moreover, genetic ablation of METTL21B function in mammalian cells caused substantial alterations in mRNA translation, as measured by ribosomal profiling. A non-canonical function for eEF1A in organization of the cellular cytoskeleton has been reported, and interestingly, METTL21B accumulated in centrosomes, in addition to the expected cytosolic localization. In summary, the present study identifies METTL21B as the enzyme responsible for methylation of eEF1A on Lys-165 and shows that this modification is dynamic, inducible and likely of regulatory importance

Lysine methylated peptides in eEF1A in wild type and Ynl024c deficient yeast.

<p>*Methylated residue indicated in bold.</p><p>**Exclusively detected in wild type cells.</p><p>Lysine methylated peptides in eEF1A in wild type and Ynl024c deficient yeast.</p

<i>In vitro</i> MTase activity of Ynl024c.

<p>Protein extract from the <i>ynl024cΔ</i> yeast strain (denoted “<i>Sc ynl024c</i>Δ”) was treated with a protein extract from <i>E</i>. <i>coli</i> either expressing 6xHis-tagged Ynl024c from the pET28a-<i>YNL024C</i> plasmid (denoted “<i>Ec</i>+ Ynl024c”) or devoid of an expression plasmid (denoted “<i>Ec</i>”), at 37°C for 60 min in the presence of [³H]AdoMet, and 1 mM GTP where indicated. Proteins were separated by SDS-PAGE, transferred to a PVDF membrane and then subjected to Ponceau S staining (bottom) and fluorograpy (top).</p

Ynl024c-mediated methylation of eEF1A <i>in vivo</i>.

<p>(A) Qualitative analysis of Ynl024c-dependent methylation of Lys390 in eEF1A. MS chromatograms gated for the different lysine methylation states of Asp-N generated peptides corresponding to aa 387–395 of <i>S</i>. <i>cerevisiae</i> eEF1A from wild-type and Ynl024c deficient (<i>ynl024cΔ</i>) yeast or these strains with overexpression of Ynl024c, denoted “Wild-type::<i>YNL024C</i>”or “<i>ynl024cΔ</i>::<i>YNL024C</i>“, respectively. The expected elution time for the relevant peptide is indicated by an arrow above the upper trace. The peak corresponding to an unrelated peptide with a m/z-ratio matching the trimethylated peptide species is indicated by an asterisk. (B) Quantitative analysis of Ynl024c-dependent methylation of Lys390 in eEF1A. Quantitative representation of the data from (A). The fractional occupancy of the various lysine methylation states was determined as the relative signal for corresponding peptides in (A), determined by integration. C-E, MS/MS fragmentation pattern supporting the identity of analysed peptides. Representative annotated spectra for un- (C), mono- (D) and trimethylated (E) peptides corresponding to peaks in (A) are shown. Spectra for the dimethylated peptide is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131426#pone.0131426.s002" target="_blank">S2 Fig</a>.</p