title: Yeast Elongator protein Elp1p does not undergo proteolytic processing in exponentially growing cells.
abstract: In eukaryotic organisms, Elongator is a six-subunit protein complex required for the formation of 5-carbamoylmethyl (ncm(5) ) and 5-methylcarboxymethyl (mcm(5) ) side chains on uridines present at the wobble position (U34 ) of tRNA. The open reading frame encoding the largest Elongator subunit Elp1p has two in-frame 5' AUG methionine codons separated by 48 nucleotides. Here, we show that the second AUG acts as the start codon of translation. Furthermore, Elp1p was previously shown to exist in two major forms of which one was generated by proteolysis of full-length Elp1p and this proteolytic cleavage was suggested to regulate Elongator complex activity. In this study, we found that the vacuolar protease Prb1p was responsible for the cleavage of Elp1p. The cleavage occurs between residues 203 (Lys) and 204 (Ala) as shown by amine reactive Tandem Mass Tag followed by LC-MS/MS (liquid chromatography mass spectrometry) analysis. However, using a modified protein extraction procedure, including trichloroacetic acid, only full-length Elp1p was observed, showing that truncation of Elp1p is an artifact occurring during protein extraction. Consequently, our results indicate that N-terminal truncation of Elp1p is not likely to regulate Elongator complex activity.
authors.affiliation: Department of Molecular Biology, Umeå University, 901 87, Umeå, Sweden.
authors.forename: Hao
authors.identifier: http://orcid.org/0000-0002-0482-597X
authors.initials: H
authors.lastname: Xu
authors.affiliation: Department of Forest Genetics and Plant Physiology, Umeå Plant Science Centre, Swedish University of Agricultural Sciences, 901 87, Umeå, Sweden.
authors.forename: Joakim
authors.initials: J
authors.lastname: Bygdell
authors.affiliation: Department of Forest Genetics and Plant Physiology, Umeå Plant Science Centre, Swedish University of Agricultural Sciences, 901 87, Umeå, Sweden.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Wingsle
authors.affiliation: Department of Molecular Biology, Umeå University, 901 87, Umeå, Sweden.
authors.forename: Anders S
authors.initials: AS
authors.lastname: Byström
mesh_terms: D000595:Amino Acid Sequence; D051548:Histone Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D010445:Peptide Elongation Factors; D029701:Saccharomyces cerevisiae Proteins; D051548:Histone Acetyltransferases; C479559:IKI3 protein, S cerevisiae
keywords: Elongator complex; Elp1p; Prb1p; Saccharomyces cerevisiae; proteolysis; tRNA modification