Elp3 and RlmN: A tale of two mitochondrial tail-anchored radical SAM enzymes in Toxoplasma gondii

Leah R. Padgett, Jenna M. Lentini, Michael J. Holmes, Krista L. Stilger, Dragony Fu, William Sullivan

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Radical S-adenosylmethionine (rSAM) enzymes use a 5’-deoxyadensyl 5’-radical to methyl-ate a wide array of diverse substrates including proteins, lipids and nucleic acids. One such enzyme, Elongator protein-3 (TgElp3), is an essential protein in Toxoplasma gondii, a protozoan parasite that can cause life-threatening opportunistic disease. Unlike Elp3 homologues which are present in all domains of life, TgElp3 localizes to the outer mitochondrial membrane (OMM) via a tail-anchored trafficking mechanism in Toxoplasma. Intriguingly, we identified a second tail-anchored rSAM domain containing protein (TgRlmN) that also localizes to the OMM. The transmembrane domain (TMD) on Toxoplasma Elp3 and RlmN homologues is required for OMM localization and has not been seen beyond the chromalveolates. Both TgElp3 and TgRlmN contain the canonical rSAM amino acid sequence motif (CxxxCxxC) necessary to form the 4Fe-4S cluster required for tRNA modifications. In E. coli, RlmN is responsible for the 2-methlyadenosine (m2A) synthesis at purine 37 in tRNA while in S. cerevisiae, Elp3 is necessary for the formation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at the wobble tRNA position. To investigate why these two rSAM enzymes localize to the mitochondrion in Toxoplasma, and whether or not TgRlmN and TgElp3 possess tRNA methyltransferase activity, a series of mutational and biochemical studies were performed. Overexpression of either TgElp3 or TgRlmN resulted in a significant parasite replication defect, but overexpression was tolerated if either the TMD or rSAM domain was mutated. Furthermore, we show the first evidence that Toxoplasma tRNAGlu contains the mcm5s2U modification, which is the putative downstream product generated by TgElp3 activity.

Original languageEnglish (US)
Article numbere0189688
JournalPLoS One
Volume13
Issue number1
DOIs
StatePublished - Jan 1 2018

Fingerprint

S-Adenosylmethionine
Toxoplasma
Toxoplasma gondii
S-adenosylmethionine
Tail
tail
Mitochondrial Membranes
Transfer RNA
Enzymes
enzymes
Membranes
Parasites
Proteins
tRNA Methyltransferases
RNA, Transfer, Glu
proteins
Amino Acid Motifs
Mitochondria
parasites
Escherichia coli

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Elp3 and RlmN : A tale of two mitochondrial tail-anchored radical SAM enzymes in Toxoplasma gondii. / Padgett, Leah R.; Lentini, Jenna M.; Holmes, Michael J.; Stilger, Krista L.; Fu, Dragony; Sullivan, William.

In: PLoS One, Vol. 13, No. 1, e0189688, 01.01.2018.

Research output: Contribution to journalArticle

Padgett, Leah R. ; Lentini, Jenna M. ; Holmes, Michael J. ; Stilger, Krista L. ; Fu, Dragony ; Sullivan, William. / Elp3 and RlmN : A tale of two mitochondrial tail-anchored radical SAM enzymes in Toxoplasma gondii. In: PLoS One. 2018 ; Vol. 13, No. 1.
@article{2e30af87b2a1422a9a8b9da6b1b7943e,
title = "Elp3 and RlmN: A tale of two mitochondrial tail-anchored radical SAM enzymes in Toxoplasma gondii",
abstract = "Radical S-adenosylmethionine (rSAM) enzymes use a 5’-deoxyadensyl 5’-radical to methyl-ate a wide array of diverse substrates including proteins, lipids and nucleic acids. One such enzyme, Elongator protein-3 (TgElp3), is an essential protein in Toxoplasma gondii, a protozoan parasite that can cause life-threatening opportunistic disease. Unlike Elp3 homologues which are present in all domains of life, TgElp3 localizes to the outer mitochondrial membrane (OMM) via a tail-anchored trafficking mechanism in Toxoplasma. Intriguingly, we identified a second tail-anchored rSAM domain containing protein (TgRlmN) that also localizes to the OMM. The transmembrane domain (TMD) on Toxoplasma Elp3 and RlmN homologues is required for OMM localization and has not been seen beyond the chromalveolates. Both TgElp3 and TgRlmN contain the canonical rSAM amino acid sequence motif (CxxxCxxC) necessary to form the 4Fe-4S cluster required for tRNA modifications. In E. coli, RlmN is responsible for the 2-methlyadenosine (m2A) synthesis at purine 37 in tRNA while in S. cerevisiae, Elp3 is necessary for the formation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at the wobble tRNA position. To investigate why these two rSAM enzymes localize to the mitochondrion in Toxoplasma, and whether or not TgRlmN and TgElp3 possess tRNA methyltransferase activity, a series of mutational and biochemical studies were performed. Overexpression of either TgElp3 or TgRlmN resulted in a significant parasite replication defect, but overexpression was tolerated if either the TMD or rSAM domain was mutated. Furthermore, we show the first evidence that Toxoplasma tRNAGlu contains the mcm5s2U modification, which is the putative downstream product generated by TgElp3 activity.",
author = "Padgett, {Leah R.} and Lentini, {Jenna M.} and Holmes, {Michael J.} and Stilger, {Krista L.} and Dragony Fu and William Sullivan",
year = "2018",
month = "1",
day = "1",
doi = "10.1371/journal.pone.0189688",
language = "English (US)",
volume = "13",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "1",

}

TY - JOUR

T1 - Elp3 and RlmN

T2 - A tale of two mitochondrial tail-anchored radical SAM enzymes in Toxoplasma gondii

AU - Padgett, Leah R.

AU - Lentini, Jenna M.

AU - Holmes, Michael J.

AU - Stilger, Krista L.

AU - Fu, Dragony

AU - Sullivan, William

PY - 2018/1/1

Y1 - 2018/1/1

N2 - Radical S-adenosylmethionine (rSAM) enzymes use a 5’-deoxyadensyl 5’-radical to methyl-ate a wide array of diverse substrates including proteins, lipids and nucleic acids. One such enzyme, Elongator protein-3 (TgElp3), is an essential protein in Toxoplasma gondii, a protozoan parasite that can cause life-threatening opportunistic disease. Unlike Elp3 homologues which are present in all domains of life, TgElp3 localizes to the outer mitochondrial membrane (OMM) via a tail-anchored trafficking mechanism in Toxoplasma. Intriguingly, we identified a second tail-anchored rSAM domain containing protein (TgRlmN) that also localizes to the OMM. The transmembrane domain (TMD) on Toxoplasma Elp3 and RlmN homologues is required for OMM localization and has not been seen beyond the chromalveolates. Both TgElp3 and TgRlmN contain the canonical rSAM amino acid sequence motif (CxxxCxxC) necessary to form the 4Fe-4S cluster required for tRNA modifications. In E. coli, RlmN is responsible for the 2-methlyadenosine (m2A) synthesis at purine 37 in tRNA while in S. cerevisiae, Elp3 is necessary for the formation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at the wobble tRNA position. To investigate why these two rSAM enzymes localize to the mitochondrion in Toxoplasma, and whether or not TgRlmN and TgElp3 possess tRNA methyltransferase activity, a series of mutational and biochemical studies were performed. Overexpression of either TgElp3 or TgRlmN resulted in a significant parasite replication defect, but overexpression was tolerated if either the TMD or rSAM domain was mutated. Furthermore, we show the first evidence that Toxoplasma tRNAGlu contains the mcm5s2U modification, which is the putative downstream product generated by TgElp3 activity.

AB - Radical S-adenosylmethionine (rSAM) enzymes use a 5’-deoxyadensyl 5’-radical to methyl-ate a wide array of diverse substrates including proteins, lipids and nucleic acids. One such enzyme, Elongator protein-3 (TgElp3), is an essential protein in Toxoplasma gondii, a protozoan parasite that can cause life-threatening opportunistic disease. Unlike Elp3 homologues which are present in all domains of life, TgElp3 localizes to the outer mitochondrial membrane (OMM) via a tail-anchored trafficking mechanism in Toxoplasma. Intriguingly, we identified a second tail-anchored rSAM domain containing protein (TgRlmN) that also localizes to the OMM. The transmembrane domain (TMD) on Toxoplasma Elp3 and RlmN homologues is required for OMM localization and has not been seen beyond the chromalveolates. Both TgElp3 and TgRlmN contain the canonical rSAM amino acid sequence motif (CxxxCxxC) necessary to form the 4Fe-4S cluster required for tRNA modifications. In E. coli, RlmN is responsible for the 2-methlyadenosine (m2A) synthesis at purine 37 in tRNA while in S. cerevisiae, Elp3 is necessary for the formation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at the wobble tRNA position. To investigate why these two rSAM enzymes localize to the mitochondrion in Toxoplasma, and whether or not TgRlmN and TgElp3 possess tRNA methyltransferase activity, a series of mutational and biochemical studies were performed. Overexpression of either TgElp3 or TgRlmN resulted in a significant parasite replication defect, but overexpression was tolerated if either the TMD or rSAM domain was mutated. Furthermore, we show the first evidence that Toxoplasma tRNAGlu contains the mcm5s2U modification, which is the putative downstream product generated by TgElp3 activity.

UR - http://www.scopus.com/inward/record.url?scp=85039931745&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85039931745&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0189688

DO - 10.1371/journal.pone.0189688

M3 - Article

C2 - 29293520

AN - SCOPUS:85039931745

VL - 13

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 1

M1 - e0189688

ER -