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Comprehensive Research Report: EFM6 (YNL024C) — A Protein-Lysine N-Methyltransferase in Saccharomyces cerevisiae

1. Gene and Protein Identity

EFM6 (Elongation Factor Methyltransferase 6) is encoded by the YNL024C open reading frame in Saccharomyces cerevisiae (strain ATCC 204508 / S288c). The protein was originally uncharacterized and was renamed from Ynl024c to Efm6 following the discovery of its enzymatic function, in accordance with the nomenclature established for similar elongation factor methyltransferases in yeast (jakobsson2015saccharomycescerevisiaeeukaryotic pages 1-2). The gene product is classified as a class I-like SAM-binding methyltransferase belonging to the seven-β-strand (7BS) methyltransferase superfamily, specifically within Methyltransferase Family 16 (MTF16), which also includes the human METTL21 proteins (jakobsson2015saccharomycescerevisiaeeukaryotic pages 5-7).

The key properties of EFM6 are summarized below:

Table: This table summarizes the core molecular, enzymatic, structural, and functional properties of the yeast EFM6/YNL024C protein based on the gathered evidence and the supplied UniProt record. It is useful as a compact reference for functional annotation and for distinguishing EFM6 from related METTL21-family methyltransferases.

2. Enzymatic Activity and Substrate Specificity

2.1 Primary Reaction

EFM6 functions as a protein-lysine N-methyltransferase (EC 2.1.1.-). Its sole identified substrate is eukaryotic translation elongation factor 1A (eEF1A), and it specifically catalyzes the methylation of lysine 390 (Lys390) on this protein using S-adenosylmethionine (AdoMet/SAM) as the methyl donor (jakobsson2015saccharomycescerevisiaeeukaryotic pages 1-2, jakobsson2015saccharomycescerevisiaeeukaryotic pages 12-14). EEF1A is a highly abundant, essential protein that functions as the eukaryotic homolog of bacterial EF-Tu, delivering aminoacylated tRNAs to the ribosomal A-site during the elongation phase of mRNA translation (jakobsson2015saccharomycescerevisiaeeukaryotic pages 2-4).

2.2 Methylation Stoichiometry and Dynamics

In wild-type yeast cells, Lys390 methylation occurs at very low occupancy, with only approximately 20% of eEF1A molecules bearing monomethylation at this residue (~0.2 methyl groups per eEF1A molecule) (jakobsson2015saccharomycescerevisiaeeukaryotic pages 12-14, jakobsson2018regulationofeukaryotic pages 7-9). In a ynl024cΔ knockout strain, Lys390 is exclusively unmethylated, confirming that Efm6 is the sole enzyme responsible for this modification (jakobsson2015saccharomycescerevisiaeeukaryotic pages 7-9). Notably, overexpression of Efm6 causes a dramatic increase in Lys390 methylation, elevating it to approximately 2 methyl groups per eEF1A molecule, with trimethylation becoming the predominant modification state (jakobsson2015saccharomycescerevisiaeeukaryotic pages 12-14, jakobsson2018regulationofeukaryotic pages 7-9). This observation indicates that the entire cellular pool of eEF1A is accessible as substrate for Efm6, and that the low in vivo methylation level reflects limited Efm6 enzyme availability under normal growth conditions rather than substrate inaccessibility (jakobsson2015saccharomycescerevisiaeeukaryotic pages 12-14).

2.3 Substrate Specificity — What EFM6 Does Not Methylate

Despite high sequence similarity to human METTL21A — an Hsp70-specific lysine methyltransferase — Efm6 does not methylate yeast Hsp70 proteins (SSA1, SSA3, SSB1, SSB2) at their corresponding lysine residues (jakobsson2015saccharomycescerevisiaeeukaryotic pages 2-4, jakobsson2015saccharomycescerevisiaeeukaryotic pages 7-9, jakobsson2015saccharomycescerevisiaeeukaryotic pages 1-2). This finding indicates that while Efm6 and METTL21A share structural features, they have diverged in substrate specificity. EFM6 was the last of the four previously known eEF1A methylation sites to have its responsible methyltransferase identified (jakobsson2015saccharomycescerevisiaeeukaryotic pages 1-2, jakobsson2015saccharomycescerevisiaeeukaryotic pages 12-14).

3. Structural Basis of Catalytic Activity

Efm6 adopts the canonical seven-β-strand (7BS) methyltransferase fold, with two additional N-terminal β-strands (z1 and z2) as revealed by structural modeling using human METTL21A as a template (jakobsson2015saccharomycescerevisiaeeukaryotic pages 5-7). The protein contains several conserved motifs characteristic of class I methyltransferases:

• Motif I, Post I, and Motif II: canonical SAM-binding motifs of the 7BS family.
• DXXY motif: a hallmark of MTF16 family enzymes, located downstream of Motif II. In Efm6, the key catalytic residues Asp170 and Tyr173 within this motif are positioned near the methyl group of AdoMet.
• Trp143: stacks with the adenine base of AdoMet, contributing to cofactor binding.
• Asp115 (in Motif Post I): forms hydrogen bonds with hydroxyl groups on the ribose moiety of AdoMet (jakobsson2015saccharomycescerevisiaeeukaryotic pages 5-7).

Sequence comparison reveals that Efm6 shows higher similarity to human METTL21A than to any other S. cerevisiae protein, including the related yeast methyltransferase Efm2 (jakobsson2015saccharomycescerevisiaeeukaryotic pages 4-5, jakobsson2015saccharomycescerevisiaeeukaryotic pages 5-7). The structural and sequence similarity between Efm6 and the METTL21 family is even higher than between some human METTL21 paralogs themselves (jakobsson2015saccharomycescerevisiaeeukaryotic pages 5-7).

4. Context Within the eEF1A Methylation Landscape

EEF1A in S. cerevisiae is one of the most extensively post-translationally modified translation factors, bearing methylation at multiple lysine residues. Five dedicated eEF1A-specific lysine methyltransferases have been identified in yeast, each targeting a distinct residue:

Table: This table summarizes the known eEF1A-specific lysine methyltransferases in S. cerevisiae, their target residues, methylation states, enzyme families, and human counterparts where known. Efm6/YNL024C is highlighted because it is the focus gene and catalyzes the Lys390 methylation event discussed in the report.

Each of these methyltransferases was identified primarily through knockout studies in S. cerevisiae, followed by mass spectrometric analysis of eEF1A methylation status (jakobsson2018regulationofeukaryotic pages 5-7, jakobsson2015saccharomycescerevisiaeeukaryotic pages 12-14). The methylation at Lys79 and Lys316 is conserved to human eEF1A, with human orthologs N6AMT2 (eEF1A-KMT1) and METTL10 (eEF1A-KMT2) catalyzing the corresponding modifications (jakobsson2018regulationofeukaryotic pages 5-7). Except for Efm1, which is a SET-domain protein, all of the identified eEF1A-specific KMTs in yeast and human belong to the 7BS family of methyltransferases (jakobsson2018regulationofeukaryotic pages 5-7).

5. Subcellular Localization

The subcellular localization of Efm6 has not been explicitly established through dedicated localization studies in the literature reviewed. However, its function can be confidently inferred to occur in the cytoplasm, as its substrate eEF1A is a cytoplasmic translation elongation factor that functions at the ribosome during mRNA translation (jakobsson2015saccharomycescerevisiaeeukaryotic pages 2-4). The closest human homolog of Efm6 discussed in this context — eEF1A-KMT3/METTL21B — has been shown to be partially cytosolic and partially localized to centrosomes, suggesting potential non-canonical functions (jakobsson2018regulationofeukaryotic pages 7-9, jakobsson2018regulationofeukaryotic pages 5-7). It has been suggested that eEF1A methylation may occur prior to complex assembly, as proteins within larger complexes may become inaccessible for methylation once assembled (jakobsson2015saccharomycescerevisiaeeukaryotic pages 14-16).

6. Biological Significance and Pathway Context

6.1 Role in Translation Regulation

EEF1A is a GTPase that delivers aminoacylated tRNAs to the ribosomal A-site during translational elongation, and its proper function is critical for efficient and accurate protein synthesis (jakobsson2015saccharomycescerevisiaeeukaryotic pages 2-4). The ablation of eEF1A-modifying methyltransferases has been associated with translation-related phenotypes such as decreased accuracy of protein synthesis and increased sensitivity to protein synthesis inhibitors (jakobsson2015saccharomycescerevisiaeeukaryotic pages 2-4). However, the specific functional consequence of Lys390 methylation by Efm6 remains largely elusive. Structural analysis of eEF1A revealed that Lys390 does not show striking co-localization with residues involved in known eEF1A functional activities, including actin interaction, nuclear transport, GTP-binding, or tRNA binding (jakobsson2015saccharomycescerevisiaeeukaryotic pages 14-16, jakobsson2015saccharomycescerevisiaeeukaryotic pages 12-14).

6.2 Hypoxia-Responsive Regulation

A particularly notable finding is that EFM6 expression is strongly upregulated by hypoxia, suggesting that Efm6-mediated Lys390 methylation may serve as a hypoxia-inducible modulator of protein synthesis (jakobsson2018regulationofeukaryotic pages 7-9). This dynamic, inducible nature of Efm6-mediated methylation is consistent with a regulatory rather than constitutive "editing" role for this modification (jakobsson2018regulationofeukaryotic pages 7-9). The concept of dynamic, stress-responsive eEF1A methylation has been extended to the mammalian system, where the close Efm6 family member eEF1A-KMT3 is induced by various stressors such as growth factor withdrawal and ER stress, with concomitant increases in methylation at its target site (jakobsson2018regulationofeukaryotic pages 5-7).

6.3 Phenotypic Effects of Loss of eEF1A Methylation

A yeast quadruple mutant in which all four previously known methylated lysine residues of eEF1A (K30R, K79R, K316R, K395R) were simultaneously replaced with arginine was viable and showed no obvious growth phenotype under standard laboratory conditions (jakobsson2018regulationofeukaryotic pages 7-9). This suggests that the effects of eEF1A lysine methylation on the basic, essential function of eEF1A are modest during near-optimal growth conditions. However, it has been proposed that methylation may protect lysine residues against ubiquitin-mediated degradation, a function that would also be preserved by lysine-to-arginine substitution, potentially masking the true importance of methylation (jakobsson2018regulationofeukaryotic pages 7-9).

6.4 The "eEF1A Code" Hypothesis

The abundance and specificity of lysine methylations on eEF1A, installed by multiple dedicated KMTs, has led to the proposal of an "eEF1A code" — analogous to the histone code — in which combinatorial lysine methylation patterns regulate eEF1A function, potentially influencing both canonical translation and non-canonical functions such as actin cytoskeleton regulation and viral replication (jakobsson2018regulationofeukaryotic pages 7-9, jakobsson2018regulationofeukaryotic pages 5-7). Efm6-mediated Lys390 methylation represents one layer of this regulatory code.

7. Evolutionary Conservation and Divergence

Efm6 is the closest S. cerevisiae sequence homolog of the human METTL21 protein family (METTL21A, METTL21B/eEF1A-KMT3, METTL21C, VCP-KMT/METTL21D), with METTL21A being the most closely related human protein in reciprocal BLAST searches (jakobsson2015saccharomycescerevisiaeeukaryotic pages 1-2, jakobsson2015saccharomycescerevisiaeeukaryotic pages 4-5). Despite this high sequence similarity, the substrate specificity has diverged: human METTL21A methylates Hsp70 proteins, whereas yeast Efm6 methylates eEF1A at Lys390 (jakobsson2015saccharomycescerevisiaeeukaryotic pages 1-2). Furthermore, the Lys390 residue in eEF1A, although structurally conserved in mammalian eEF1A (corresponding to Lys392), is exclusively unmethylated in rabbit reticulocytes and human HeLa cells, indicating that this particular methylation event is yeast-specific (jakobsson2015saccharomycescerevisiaeeukaryotic pages 12-14). This represents a case of functional divergence following gene duplication and speciation, where homologous enzymes have acquired different substrate specificities.

8. Summary

EFM6 (YNL024C, UniProt P53970) is a seven-β-strand methyltransferase in S. cerevisiae that catalyzes the SAM-dependent monomethylation of eEF1A at Lys390. It was identified in 2015 as the last of the yeast eEF1A-specific lysine methyltransferases (jakobsson2015saccharomycescerevisiaeeukaryotic pages 1-2). The enzyme operates in the cytoplasm, where its substrate eEF1A functions in translational elongation. Its methylation activity is notable for being low-occupancy under normal conditions but highly inducible through enzyme overexpression and under hypoxic stress, suggesting a regulatory rather than constitutive role (jakobsson2018regulationofeukaryotic pages 7-9). Although structurally homologous to human METTL21 proteins, Efm6 targets eEF1A rather than Hsp70 proteins, and the Lys390 methylation event it catalyzes is not conserved in mammals (jakobsson2015saccharomycescerevisiaeeukaryotic pages 12-14, jakobsson2015saccharomycescerevisiaeeukaryotic pages 1-2). The precise functional consequence of Lys390 methylation remains an active area of investigation, but current evidence places it within a broader regulatory framework — the proposed "eEF1A code" — that modulates translation through combinatorial post-translational modifications of this essential elongation factor.

References

1. (jakobsson2015saccharomycescerevisiaeeukaryotic pages 1-2): Magnus E. Jakobsson, Erna Davydova, Jędrzej Małecki, Anders Moen, and Pål Ø. Falnes. Saccharomyces cerevisiae eukaryotic elongation factor 1a (eef1a) is methylated at lys-390 by a mettl21-like methyltransferase. PLoS ONE, 10:e0131426, Jun 2015. URL: https://doi.org/10.1371/journal.pone.0131426, doi:10.1371/journal.pone.0131426. This article has 45 citations and is from a peer-reviewed journal.

2. (jakobsson2015saccharomycescerevisiaeeukaryotic pages 5-7): Magnus E. Jakobsson, Erna Davydova, Jędrzej Małecki, Anders Moen, and Pål Ø. Falnes. Saccharomyces cerevisiae eukaryotic elongation factor 1a (eef1a) is methylated at lys-390 by a mettl21-like methyltransferase. PLoS ONE, 10:e0131426, Jun 2015. URL: https://doi.org/10.1371/journal.pone.0131426, doi:10.1371/journal.pone.0131426. This article has 45 citations and is from a peer-reviewed journal.

3. (jakobsson2015saccharomycescerevisiaeeukaryotic pages 12-14): Magnus E. Jakobsson, Erna Davydova, Jędrzej Małecki, Anders Moen, and Pål Ø. Falnes. Saccharomyces cerevisiae eukaryotic elongation factor 1a (eef1a) is methylated at lys-390 by a mettl21-like methyltransferase. PLoS ONE, 10:e0131426, Jun 2015. URL: https://doi.org/10.1371/journal.pone.0131426, doi:10.1371/journal.pone.0131426. This article has 45 citations and is from a peer-reviewed journal.

4. (jakobsson2018regulationofeukaryotic pages 5-7): Magnus E. Jakobsson, Jędrzej Małecki, and Pål Ø. Falnes. Regulation of eukaryotic elongation factor 1 alpha (eef1a) by dynamic lysine methylation. RNA Biology, 15:314-319, Mar 2018. URL: https://doi.org/10.1080/15476286.2018.1440875, doi:10.1080/15476286.2018.1440875. This article has 64 citations and is from a peer-reviewed journal.

5. (jakobsson2018regulationofeukaryotic pages 7-9): Magnus E. Jakobsson, Jędrzej Małecki, and Pål Ø. Falnes. Regulation of eukaryotic elongation factor 1 alpha (eef1a) by dynamic lysine methylation. RNA Biology, 15:314-319, Mar 2018. URL: https://doi.org/10.1080/15476286.2018.1440875, doi:10.1080/15476286.2018.1440875. This article has 64 citations and is from a peer-reviewed journal.

6. (jakobsson2015saccharomycescerevisiaeeukaryotic pages 2-4): Magnus E. Jakobsson, Erna Davydova, Jędrzej Małecki, Anders Moen, and Pål Ø. Falnes. Saccharomyces cerevisiae eukaryotic elongation factor 1a (eef1a) is methylated at lys-390 by a mettl21-like methyltransferase. PLoS ONE, 10:e0131426, Jun 2015. URL: https://doi.org/10.1371/journal.pone.0131426, doi:10.1371/journal.pone.0131426. This article has 45 citations and is from a peer-reviewed journal.

7. (jakobsson2015saccharomycescerevisiaeeukaryotic pages 4-5): Magnus E. Jakobsson, Erna Davydova, Jędrzej Małecki, Anders Moen, and Pål Ø. Falnes. Saccharomyces cerevisiae eukaryotic elongation factor 1a (eef1a) is methylated at lys-390 by a mettl21-like methyltransferase. PLoS ONE, 10:e0131426, Jun 2015. URL: https://doi.org/10.1371/journal.pone.0131426, doi:10.1371/journal.pone.0131426. This article has 45 citations and is from a peer-reviewed journal.

8. (jakobsson2015saccharomycescerevisiaeeukaryotic pages 7-9): Magnus E. Jakobsson, Erna Davydova, Jędrzej Małecki, Anders Moen, and Pål Ø. Falnes. Saccharomyces cerevisiae eukaryotic elongation factor 1a (eef1a) is methylated at lys-390 by a mettl21-like methyltransferase. PLoS ONE, 10:e0131426, Jun 2015. URL: https://doi.org/10.1371/journal.pone.0131426, doi:10.1371/journal.pone.0131426. This article has 45 citations and is from a peer-reviewed journal.

9. (jakobsson2015saccharomycescerevisiaeeukaryotic pages 14-16): Magnus E. Jakobsson, Erna Davydova, Jędrzej Małecki, Anders Moen, and Pål Ø. Falnes. Saccharomyces cerevisiae eukaryotic elongation factor 1a (eef1a) is methylated at lys-390 by a mettl21-like methyltransferase. PLoS ONE, 10:e0131426, Jun 2015. URL: https://doi.org/10.1371/journal.pone.0131426, doi:10.1371/journal.pone.0131426. This article has 45 citations and is from a peer-reviewed journal.

Artifacts

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Citations

1. jakobsson2015saccharomycescerevisiaeeukaryotic pages 1-2
2. jakobsson2015saccharomycescerevisiaeeukaryotic pages 5-7
3. jakobsson2015saccharomycescerevisiaeeukaryotic pages 2-4
4. jakobsson2015saccharomycescerevisiaeeukaryotic pages 7-9
5. jakobsson2015saccharomycescerevisiaeeukaryotic pages 12-14
6. jakobsson2018regulationofeukaryotic pages 5-7
7. jakobsson2015saccharomycescerevisiaeeukaryotic pages 14-16
8. jakobsson2018regulationofeukaryotic pages 7-9
9. jakobsson2015saccharomycescerevisiaeeukaryotic pages 4-5
10. https://doi.org/10.1371/journal.pone.0131426,
11. https://doi.org/10.1080/15476286.2018.1440875,