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Importance of individual amino acids in the Switch I region in eEF2 studied by functional complementation in S. cerevisiae
Södertörn University, School of Life Sciences. Stockholm University.
Södertörn University, School of Life Sciences.
2008 (English)In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 90, no 5, 736-748 p.Article in journal (Refereed) Published
Abstract [en]

Elongation factor 2 (eEF2) is a member of the G-protein super family. G-proteins undergo conformational changes associated with binding of the guanosine nucleotide and hydrolysis of the bound GTP. These structural rearrangements affects the Switch I region (also known as the Effector loop). We have studied the role of individual amino acids in the Switch I region (amino acids 25-73) of S. cerevisiae eEF2 using functional complementation in yeast. 21 point mutations in the Switch I region were created by site-directed mutagenesis. Mutants K49R, E52Q, A53G, F55Y, K60R, Q63A, T68S, 169M and A73G were functional while mutants R54H, F55N, D57A, D57E, D57S, R59K, R59M, Q63E, R65A, R65N, T68A and T68M were inactive. Expression of mutants K49R, A53G, Q63A, 169M and A73G was associated with markedly decreased growth rates and yeast cells expressing mutants A53G and 169M became temperature sensitive. The functional capacity of eEF2 in which the major part Switch I (amino acids T56 to 169) was converted into the homologous sequence found in EF-G from E. coli was also studied. This protein chimera could functionally replace yeast eEF2 in vivo. Yeast cells expressing this mutant grew extremely slowly, showed increased cell death and became temperature sensitive. The ability of the mutant to replace authentic eEF2 in vivo indicates that the structural rearrangement of Switch I necessary for eEF2 function is similar in eukaryotes and bacteria. The effect of two point mutations in the P-loop was also studied. Mutant A25G but not A25V could functionally replace yeast eEF2 even if cells expressing the mutant grew slowly. The A25G mutation converted the consensus sequences AXXXXGK[T/S] in eEF2 to the corresponding motif GXXXXGK[T/S] found in all other G-proteins, suggesting that the alanine found in the P-loop of peptidyltranslocases are not essential for function.

Place, publisher, year, edition, pages
2008. Vol. 90, no 5, 736-748 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:sh:diva-14152DOI: 10.1016/j.biochi.2008.01.006ISI: 000256256000007PubMedID: 18267126Scopus ID: 2-s2.0-43049110270OAI: oai:DiVA.org:sh-14152DiVA: diva2:467023
Available from: 2011-12-18 Created: 2011-12-16 Last updated: 2017-02-13Bibliographically approved
In thesis
1. Elongation factor 2: A key component of the translation machinery in eukaryotes: Properties of yeast elongation factor 2 studied in vivo
Open this publication in new window or tab >>Elongation factor 2: A key component of the translation machinery in eukaryotes: Properties of yeast elongation factor 2 studied in vivo
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
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.

Place, publisher, year, edition, pages
Stockholm: Wenner-Gren Institute for Experimental Biology, Stockholm university, 2008. 56 p.
Keyword
Elongation factor 2, yeast, ribosome, phosphorylation, Switch I, site-directed mutagenesis, functional complementation
National Category
Cell Biology
Identifiers
urn:nbn:se:sh:diva-32038 (URN)978-91-7155-634-9 (ISBN)
Public defence
2008-06-04, MA331, Alfred Nobels allé 7, Huddinge, 13:00 (English)
Opponent
Supervisors
Available from: 2017-02-13 Created: 2017-02-13 Last updated: 2017-02-13Bibliographically approved

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