Mannosyl-diinositolphospho-ceramide, the major yeast plasma membrane sphingolipid, governs toxicity of <em>Kluyveromyces lactis</em> zymocin

Kluyveromyces lactis zymocin, a trimeric (αβγ) protein toxin complex, inhibits proliferation of Saccharomyces cerevisiae cells. Here we present an analysis of kti6 mutants, which resist exogenous zymocin but are sensitive to intracellular expression of its inhibitory γ-toxin subunit, suggesting that KTI6 encodes a factor needed for toxin entry into the cell. Consistent with altered cell surface properties, kti6 cells resist hygromycin B, syringomycin E, and nystatin, antibiotics that require intact membrane potentials or provoke membrane disruption. KTI6 is allelic to IPT1, coding for mannosyl-diinositolphospho-ceramide [M(IP)(2)C] synthase, which produces M(IP)(2)C, the major plasma membrane sphingolipid. kti6 membranes lack M(IP)(2)C and sphingolipid mutants that have reduced levels of M(IP)(2)C precursors, including the sphingolipid building block ceramide survive zymocin. In addition, kti6/ipt1 cells allow zymocin docking but prevent import of its toxic γ-subunit. Genetic analysis indicates that Kti6 is likely to act upstream of lipid raft proton pump Kti10/Pma1, a previously identified zymocin sensitivity factor. In sum, M(IP)(2)C operates in a plasma membrane step that follows recognition of cell wall chitin by zymocin but precedes the involvement of elongator, the potential toxin target

Elongator Complex Influences Telomeric Gene Silencing and DNA Damage Response by Its Role in Wobble Uridine tRNA Modification

Elongator complex is required for formation of the side chains at position 5 of modified nucleosides 5-carbamoylmethyluridine (ncm(5)U(34)), 5-methoxycarbonylmethyluridine (mcm(5)U(34)), and 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U(34)) at wobble position in tRNA. These modified nucleosides are important for efficient decoding during translation. In a recent publication, Elongator complex was implicated to participate in telomeric gene silencing and DNA damage response by interacting with proliferating cell nuclear antigen (PCNA). Here we show that elevated levels of tRNA(Lys) (s(2) ) (UUU), tRNA(Gln) (s(2) ) (UUG), and tRNA(Glu) (s(2) ) (UUC), which in a wild-type background contain the mcm(5)s(2)U nucleoside at position 34, suppress the defects in telomeric gene silencing and DNA damage response observed in the Elongator mutants. We also found that the reported differences in telomeric gene silencing and DNA damage response of various elp3 alleles correlated with the levels of modified nucleosides at U(34). Defects in telomeric gene silencing and DNA damage response are also observed in strains with the tuc2Δ mutation, which abolish the formation of the 2-thio group of the mcm(5)s(2)U nucleoside in tRNA(Lys) (mcm(5) (s(2) ) (UUU) ), tRNA(Gln) (mcm(5) (s(2) ) (UUG) ), and tRNA(Glu) (mcm(5) (s(2) ) (UUC) ). These observations show that Elongator complex does not directly participate in telomeric gene silencing and DNA damage response, but rather that modified nucleosides at U(34) are important for efficient expression of gene products involved in these processes. Consistent with this notion, we found that expression of Sir4, a silent information regulator required for assembly of silent chromatin at telomeres, was decreased in the elp3Δ mutants

Structural basis for tRNA modification by Elp3 from Dehalococcoides mccartyi

International audienceDuring translation elongation, decoding is based on the recognition of codons by corresponding tRNA anticodon triplets. Molecular mechanisms that regulate global protein synthesis via specific base modifications in tRNA anticodons are receiving increasing attention. The conserved eukaryotic Elongator complex specifically modifies uridines located in the wobble base position of tRNAs. Mutations in Elongator subunits are associated with certain neurodegenerative diseases and cancer. Here we present the crystal structure of D. mccartyi Elp3 (DmcElp3) at 2.15-Å resolution. Our results reveal an unexpected arrangement of Elp3 lysine acetyltransferase (KAT) and radical S-adenosyl methionine (SAM) domains, which share a large interface and form a composite active site and tRNA-binding pocket, with an iron–sulfur cluster located in the dimerization interface of two DmcElp3 molecules. Structure-guided mutagenesis studies of yeast Elp3 confirmed the relevance of our findings for eukaryotic Elp3s and should aid in understanding the cellular functions and pathophysiological roles of Elongator