Sequence Requirement for Trimethylation of Yeast Cytochrome<i>c</i>

Takakura, Hikaru; Yamamoto, Tetsuro; Sherman, Fred

doi:10.1021/bi962245n

Cited by 14 publications

(12 citation statements)

References 37 publications

(39 reference statements)

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“…The lack of methylation at any site other than Lys-72 is consistent with the results of Takakura et al (32), who investigated the amino acid sequence requirement for trimethylation of Lys-72 by examining 21 altered iso-1-cytochromes c having single replacements in the region encompassing residues 67 through 77. Their study revealed that tyrosine 74 is critical for trimethylation of Lys-72, whereas replacements at other positions did not produce significant diminutions.…”

Section: Ctm1p Is a Methyltransferase That Specifically Acts On Cytocsupporting

confidence: 87%

Cytochrome c Methyltransferase, Ctm1p, of Yeast

Polevoda

Martzen

Das

et al. 2000

Journal of Biological Chemistry

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Cytochromes c from plants and fungi, but not higher animals, contain methylated lysine residues at specific positions, including for example, the trimethylated lysine at position 72 in iso-1-cytochrome c of the yeast Saccharomyces cerevisiae. Testing of 6,144 strains of S. cerevisiae, each overproducing a different open reading frame fused to glutathione S-transferase, previously revealed that YHR109w was associated with an activity that methylated horse cytochrome c. We show here that this open reading frame, denoted Ctm1p, is specifically responsible for trimethylating lysine 72 of iso-1-cytochrome c. Unmethylated forms of cytochrome c but not other proteins or nucleic acids are methylated in vitro by Ctm1p produced in S. cerevisiae or Escherichia coli. Iso-1-cytochrome c purified from a ctm1-⌬ strain is not trimethylated in vivo, whereas the K72R mutant form, or the trimethylated Lys-72 form of iso-1-cytochrome c, are not significantly methylated by Ctm1p in vitro. Like apocytochrome c, but in contrast to holocytochrome c, Ctm lp is located in the cytosol, consistent with the view that the natural substrate is apocytochrome c. The ctm1-⌬ strain lacking the methyltransferase did not exhibit any growth defect on a variety of media and growth conditions, and the unmethylated iso-1-cytochrome c was produced at the normal level and exhibited the normal activity in vivo. Ctm1p and cytochrome c were coordinately regulated during anaerobic to aerobic transition, a finding consistent with the view that this methyltransferase evolved to act on cytochrome c.

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Section: Ctm1p Is a Methyltransferase That Specifically Acts On Cytocsupporting

confidence: 87%

Cytochrome c Methyltransferase, Ctm1p, of Yeast

Polevoda

Martzen

Das

et al. 2000

Journal of Biological Chemistry

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“…Despite findings that trimethylation does affect the molecule in subtle ways (22,30), the biological reason for retaining the methylase through evolution in yeast has remained elusive (18,19). Our finding that trimethylation blocks pro-apoptotic activity initially promised a resolution to this issue.…”

Section: Discussionmentioning

confidence: 87%

“…Specific lysine residues are methylated in cytochromes c from certain eukaryotes, including fungi, plants, and some protozoa, but not in animals (18,19). Cytochromes c from many fungi, including Saccharomyces cerevisiae, contain trimethyllysine at the single position 72 (20).…”

mentioning

confidence: 99%

“…It is believed that apocytochrome c is trimethylated co-or post-translationally by a specific methyltransferase before uptake by mitochondria (21,22). In fact, vertebrate cytochromes c are at least partially trimethylated when expressed in yeast (16,19). The biological function and importance of cytochrome c methylation is unknown, and mutant forms of iso-1-cytochrome c lacking trimethyllysine 72 retain nearly full activity in vivo (19,23).…”

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confidence: 99%

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Determinants of Cytochrome c Pro-apoptotic Activity

Kluck¹,

Ellerby²,

Ellerby³

et al. 2000

Journal of Biological Chemistry

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113

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Cytochrome c released from vertebrate mitochondria engages apoptosis by triggering caspase activation. We previously reported that, whereas cytochromes c from higher eukaryotes can activate caspases in Xenopus egg and mammalian cytosols, iso-1 and iso-2 cytochromes c from the yeast Saccharomyces cerevisiae cannot. Here we examine whether the inactivity of the yeast isoforms is related to a post-translational modification of lysine 72, N-⑀-trimethylation. This modification was found to abrogate pro-apoptotic activity of metazoan cytochrome c expressed in yeast. However, iso-1 cytochrome c lacking the trimethylation modification also was devoid of pro-apoptotic activity. Thus, both lysine 72 trimethylation and other features of the iso-1 sequence preclude pro-apoptotic activity. Competition studies suggest that the lack of pro-apoptotic activity was associated with a low affinity for Apaf-1. As cytochromes c that lack apoptotic function still support respiration, different mechanisms appear to be involved in the two activities.

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“…These considerations suggest that the SET domain methyltransferase Efm1 modifies lysine 390, although this needs to be experimentally confirmed. It is important to note that the overall native structure of at least one substrate protein for SET domain methyltransferases has been observed to be required for substrate recognition and this may be a determining factor rather than, or in addition to, the primary sequence [31]. …”

Section: Resultsmentioning

confidence: 99%

Two novel methyltransferases acting upon eukaryotic elongation factor 1A in Saccharomyces cerevisiae

Lipson¹,

Webb²,

Clarke³

2010

Archives of Biochemistry and Biophysics

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Eukaryotic elongation factor 1A (eEF1A1) is an abundant cytosolic protein in Saccharomyces cerevisiae and is well conserved amongst species. This protein undergoes multiple posttranslational modifications, including the N-methylation of four side chain lysine residues. However, the enzyme(s) responsible for catalyzing these modifications have remained elusive. Here we show by intact protein mass spectrometry that deletion of either of two genes coding for putative methyltransferases results in a loss in mass of eEF1A. Deletion of the YHL039W gene, a member of the SET domain subfamily including cytochrome c and ribosomal protein lysine methyltransferases, results in an eEF1A mass loss corresponding to a single methyl group. Deletion in the YIL064W/SEE1 gene, encoding a well conserved seven beta strand methyltransferase sequence, has been shown previously to affect vesicle transport; in this work we show that deletion results in the loss of two methyl group equivalents from eEF1A. We find that deletion of thirty five other putative and established SET domain and seven beta strand methyltransferases has no effect on the mass of eEF1A. Finally, we show that wild type extracts, but not YIL064W/SEE1 mutant extracts, can catalyze the S-adenosylmethionine-dependent in vitro methylation of hypomethylated eEF1A. We suggest that YHL039W (now designated EFM1 for elongation factor methyltransferase 1) and YIL064W/SEE1 encode distinct eEF1A methyltransferases that respectively monomethylate and dimethylate this protein at lysine residues.

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Sequence Requirement for Trimethylation of Yeast Cytochromec

Cited by 14 publications

References 37 publications

Cytochrome c Methyltransferase, Ctm1p, of Yeast

Cytochrome c Methyltransferase, Ctm1p, of Yeast

Determinants of Cytochrome c Pro-apoptotic Activity

Two novel methyltransferases acting upon eukaryotic elongation factor 1A in Saccharomyces cerevisiae

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