Yeast Elongator protein Elp1p does not undergo proteolytic processing in exponentially growing cells

MicrobiologyOpen

Hao Xu¹  Joakim Bygdell²  Gunnar Wingsle² 

[1]ORCID:orcid.org/0000-0002-0482-597X

[2]Department of Forest Genetics and Plant Physiology, Umeå Plant Science Centre, Swedish University of Agricultural Sciences, Umeå, Sweden

关键词: Elongator complex;    Elp1p;    Prb1p;    proteolysis;    Saccharomyces cerevisiae;    tRNA modification;   

DOI  :  10.1002/mbo3.285

来源: Wiley

【 摘 要 】

Abstract

In eukaryotic organisms, Elongator is a six-subunit protein complex required for the formation of 5-carbamoylmethyl (ncm⁵) and 5-methylcarboxymethyl (mcm⁵) 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.

【 授权许可】

CC BY   
© 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd

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