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

Hao Xu,Joakim Bygdell,Anders S Byström,Gunnar Wingsle

doi:10.1002/mbo3.285

Hao Xu, Joakim Bygdell + Show 2 more

Open Access

https://doi.org/10.1002/mbo3.285

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Journal: MicrobiologyOpen	Publication Date: Sep 25, 2015
Citations: 25	License type: CC BY 4.0

Affiliation: Umeå University, Umeå Plant Science Centre

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.

Highlights

The Elongator complex of Saccharomyces cerevisiae was first reported to be associated with the hyper phosphorylated elongating form of RNA polymerase II (Pol II) and three proteins (Elp1p, Elp2p, and Elp3p) constituted the identified complex (Otero et al 1999)
Translation of Elp1p starts at the second AUG of the ELP1 open reading frame (ORF)
When ATG1 was mutated to TTG, the expression level of Elp1p was similar as wild type, whereas when

Summary

Introduction

The Elongator complex of Saccharomyces cerevisiae was first reported to be associated with the hyper phosphorylated elongating form of RNA polymerase II (Pol II) and three proteins (Elp1p, Elp2p, and Elp3p) constituted the identified complex (Otero et al 1999). Loss of Urm1p or Kti11p increases the amount of truncated Elp1p, abolishes formation of mcm or s2 group of mcm5s2U34, and makes cells resistant to γ-toxin, suggesting that both Kti11p and Urm1p influenced Elp1p proteolysis and are required for proper Elongator function/ regulation (Fichtner et al 2003). It was not established how the truncated form of Elp1p was generated, neither was the exact truncation site determined. We found that appearance of N-terminal truncated Elp1p is a preparation artifact which can be circumvented using an alternative protein extraction method

Experimental Procedures

Results and Discussion

A R Tryptic peptide