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Amino acids Thr56 and Thr58 are not essential for elongation factor 2 function in yeast
Södertörns högskola, Institutionen för livsvetenskaper. Stockholm University.
Södertörns högskola, Institutionen för livsvetenskaper. Stockholm University.
Södertörns högskola, Institutionen för livsvetenskaper.
2007 (Engelska)Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, nr 20, s. 5285-5297Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Yeast elongation factor 2 is an essential protein that contains two highly conserved threonine residues, T56 and T58, that could potentially be phosphorylated by the Rck2 kinase in response to environmental stress. The importance of residues T56 and T58 for elongation factor 2 function in yeast was studied using site directed mutagenesis and functional complementation. Mutations T56D, T56G, T56K, T56N and T56V resulted in nonfunctional elongation factor 2 whereas mutated factor carrying point mutations T56M, T56C, T56S, T58S and T58V was functional. Expression of mutants T56C, T56S and T58S was associated with reduced growth rate. The double mutants T56M/T58W and T56M/T58V were also functional but the latter mutant caused increased cell death and considerably reduced growth rate. The results suggest that the physiological role of T56 and T58 as phosphorylation targets is of little importance in yeast under standard growth conditions. Yeast cells expressing mutants T56C and T56S were less able to cope with environmental stress induced by increased growth temperatures. Similarly, cells expressing mutants T56M and T56M/T58W were less capable of adapting to increased osmolarity whereas cells expressing mutant T58V behaved normally. All mutants tested were retained their ability to bind to ribosomes in vivo. However, mutants T56D, T56G and T56K were under-represented on the ribosome, suggesting that these nonfunctional forms of elongation factor 2 were less capable of competing with wild-type elongation factor 2 in ribosome binding. The presence of nonfunctional but ribosome binding forms of elongation factor 2 did not affect the growth rate of yeast cells also expressing wild-type elongation factor 2.

Ort, förlag, år, upplaga, sidor
2007. Vol. 274, nr 20, s. 5285-5297
Nationell ämneskategori
Cellbiologi
Identifikatorer
URN: urn:nbn:se:sh:diva-14205DOI: 10.1111/j.1742-4658.2007.06054.xISI: 000249882400009PubMedID: 17892487Scopus ID: 2-s2.0-34848907218OAI: oai:DiVA.org:sh-14205DiVA, id: diva2:467959
Tillgänglig från: 2011-12-20 Skapad: 2011-12-19 Senast uppdaterad: 2017-12-08Bibliografiskt granskad
Ingår i avhandling
1. Elongation factor 2: A key component of the translation machinery in eukaryotes: Properties of yeast elongation factor 2 studied in vivo
Öppna denna publikation i ny flik eller fönster >>Elongation factor 2: A key component of the translation machinery in eukaryotes: Properties of yeast elongation factor 2 studied in vivo
2008 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Synthesis of proteins is performed by the ribosome, a large ribonucleoprotein complex. Apart from the ribosome, numerous protein factors participate in this process. Elongation factor 2 (eEF2) is one of these factors. eEF2 is an essential protein with a mol. mass of about 100 kDa. The amino acid sequence of eEF2 is highly conserved in different organisms. eEF2 from S. cerevisiae contains 842 amino acids. The role of eEF2 in protein synthesis is to participate in the translocation of tRNAs from the A- and P-sites on the ribosome to the P- and E-sites. This movement of tRNAs is accompanied by a simultaneous movement of mRNA by one codon. eEF2 consists of six domains referred to as domains G, G′ and II-V, belongs to the G-protein super-family and possesses all structural motifs characterizing proteins in this family. eEF2 binds to the ribosome in complex with GTP. After GTP hydrolysis and translocation, it leaves the ribosome bound to GDP. The rate of protein synthesis in the cell can be regulated by phosphorylation of eEF2. Phosphorylation occurs on two threonine residues, situated in the G domain of the factor. Phosphorylation of eEF2 is catalysed by Rck2-kinase in yeast which is activated in response to osmotic stress. Despite the high degree of conservation of the threonine residues, they are not essential for yeast cell under normal growth conditions. However, under mild osmotic stress the growth rate of the cells lacking threonine residues was decreased. Region where threonine residues are located, called Switch I. Cryo-EM reconstruction shows that this region adopts ordered conformation when the eEF2•GTP complex is bound to the ribosome but became structurally disordered upon GTP hydrolysis. Mutagenesis of individual amino acids in Switch I resulted in both functional and non-functional eEF2 depending on the site of mutation and the substituting amino acid. Both functional and non-functional Switch I mutants were able to bind to the ribosome, indicating that mutations did not abolish the capacity of the factor to bind GTP. Yeast eEF2 with Switch I region from E. coli was able to substitute the wild type protein in vivo, though the growth rate of these cells was severely impaired. The eEF2-dependent GTP hydrolysis can be activated by ribosome from heterologous sources as seen in vitro. However, eEF2 from A. thaliana, D. melanogaster and S. solfataricus could not substi-tute yeast eEF2 in vivo. This may indicate additional roles of eEF2 in the yeast cell, apart from translocation itself.

Ort, förlag, år, upplaga, sidor
Stockholm: Wenner-Gren Institute for Experimental Biology, Stockholm university, 2008. s. 56
Nyckelord
Elongation factor 2, yeast, ribosome, phosphorylation, Switch I, site-directed mutagenesis, functional complementation
Nationell ämneskategori
Cellbiologi
Identifikatorer
urn:nbn:se:sh:diva-32038 (URN)978-91-7155-634-9 (ISBN)
Disputation
2008-06-04, MA331, Alfred Nobels allé 7, Huddinge, 13:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2017-02-13 Skapad: 2017-02-13 Senast uppdaterad: 2017-02-13Bibliografiskt granskad

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Bartish, GalynaMoradi, HosseinNygård, Odd

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