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Ribosomal proteins L5, L15 and elongation factor 2, three vital components of the translation machinery: Functional features of RPL5, RPL15 and EF2 from Saccharomyces cerevisiae studied in vivo
Stockholm University, Faculty of Science, The Wenner-Gren Institute . (Roger Karlsson)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein synthesis is an essential, energy consuming and tightly regulated process in all living cells. A central core of the cellular protein-factory is a macromolecule called ribosome. Ribosomes are composed of ribosomal RNAs (rRNAs) and proteins (RPs). Additional components such as elongation factors (EFs) also contribute to this process. rRNAs are known as the catalytic constituents of the ribosome, while RPs are regarded as scaffold of the rRNA backbone. Despite this common view, in recent years, the functional importance of RPs has become more evident. In addition, RPs also carry extra-ribosomal functions some of which are linked to various diseases.

In the current thesis I have attempted to highlight the importance of the structural features of two ribosomal proteins, YrpL5 and YrpL15A, present in the large ribosomal subunit and YeEF2. The results presented here are based on mutagenesis analysis, combined with functional complementation approach in the baker’s yeast S.cerevisiae.

Introduced mutations show various degrees of cellular effects; YrpL15A tolerated inserted mutations greater than YrpL5. Nevertheless, YrpL15A proved to be more sensitive in its terminal-ends. This is presumably due to close contacts to the neighbouring molecules through these regions. On the other hand the N-terminal of YrpL5 displays a more permissive character for introduced mutations. In addition, A.thaliana orthologue to rpL15 could functionally substitute for yeast rpL15A. In contrast orthologues of rpL5 from A.thaliana, D.melanogaster and M. musculus were unable to functionally substitute for yeast rpL5. This could be an indication of species-specific features in YrpL5. Furthermore, two regulatory and highly conserved amino acids Thr56 and Thr58 displayed unessential functional role in yeast eEF2 under standard growth conditions. However, they showed to be important for YeEF2 function under mild osmotic stress. This may point to alternative regulatory mechanism for YeEF2.

Place, publisher, year, edition, pages
Stockholm: The Wenner-Gren Institute, Stockholm University , 2011. , 58 p.
Keyword [en]
Ribosome, Ribosomal protein, Elongation factor, Translation
National Category
Cell Biology
Research subject
Cell Biology
Identifiers
URN: urn:nbn:se:su:diva-63520ISBN: 978-91-7447-378-0 (print)OAI: oai:DiVA.org:su-63520DiVA: diva2:450868
Public defence
2011-11-25, lecture room E306, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2011-11-02 Created: 2011-10-21 Last updated: 2011-10-26Bibliographically approved
List of papers
1. Functional features of the C-terminal region of yeast ribosomal protein L5
Open this publication in new window or tab >>Functional features of the C-terminal region of yeast ribosomal protein L5
2008 (English)In: Molecular Genetics and Genomics, ISSN 1617-4615, E-ISSN 1617-4623, Vol. 280, no 4, 337-350 p.Article in journal (Refereed) Published
Abstract [en]

The aim of this study was to analyze the functional importance of the C-terminus of the essential yeast ribosomal protein L5 (YrpL5). Previous studies have indicated that the C-terminal region of YrpL5 forms an α-helix with a positively charged surface that is involved in protein–5S rRNA interaction. Formation of an YrpL5·5S rRNA complex is a prerequisite for nuclear import of YrpL5. Here we have tested the importance of the α-helix and the positively charged surface for YrpL5 function in Saccharomyces cerevisiae using site directed mutagenesis in combination with functional complementation. Alterations in the sequence forming the putative α-helix affected the functional capacity of YrpL5. However, the effect did not correlate with a decreased ability of the protein to bind to 5S rRNA as all rpL5 mutants tested were imported to the nucleus whether or not the α-helix or the positively charged surface were intact. The alterations introduced in the C-terminal sequence affected the growth rate of cells expressing mutant but functional forms of YrpL5. The reduced growth rate was correlated with a reduced ribosomal content per cell indicating that the alterations introduced in the C-terminus interfered with ribosome assembly.

Keyword
Functional complementation, Mutation analysis, Ribosomal protein L5, S. cerevisiae
National Category
Cell Biology
Research subject
Cellbiology
Identifiers
urn:nbn:se:su:diva-25114 (URN)10.1007/s00438-008-0369-7 (DOI)000258902300007 ()
Available from: 2008-05-13 Created: 2008-05-12 Last updated: 2017-12-13Bibliographically approved
2. Amino acids Thr56 and Thr58 are not essential for elongation factor 2 function in yeast
Open this publication in new window or tab >>Amino acids Thr56 and Thr58 are not essential for elongation factor 2 function in yeast
2007 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, no 20, 5285-5297 p.Article in journal (Refereed) 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.

Keyword
elongation factor 2, functional complementation, osmostress, phosphorylation, yeast
National Category
Cell Biology
Research subject
Cellbiology
Identifiers
urn:nbn:se:su:diva-25111 (URN)10.1111/j.1742-4658.2007.06054.x (DOI)000249882400009 ()
Available from: 2008-05-13 Created: 2008-05-12 Last updated: 2017-12-13Bibliographically approved
3. Functional characterization of ribosomal protein L15 from Saccaromyces cerevisiae
Open this publication in new window or tab >>Functional characterization of ribosomal protein L15 from Saccaromyces cerevisiae
2009 (English)In: Current Genetics, ISSN 0172-8083, E-ISSN 1432-0983, Vol. 55, no 2, 111-125 p.Article in journal (Refereed) Published
Abstract [en]

In this study we provide general information on the little studied eukaryotic ribosomal protein rpL15. Saccharomyces cerevisiae has two genes, YRPL15A and YRPL15B that could potentially code for yeast rpL15 (YrpL15). YRPL15A is essential while YRPL15B is dispensable. However, a plasmid-borne copy of the YRPL15B gene, controlled by the GAL1 promoter or by the promoter controlling expression of the YRPL15A gene, can functionally complement YrpL15A in yeast cells, while the same gene controlled by the authentic promoter is inactive. Analysis of the levels of YrpL15B-mRNA in yeast cells shows that the YRPL15B gene is inactive in transcription. The function of YrpL15A is highly resilient to single and multiple amino acid substitutions. In addition, minor deletions from both the N- and C-terminal ends of YrpL15A has no effect on protein function, while addition of a C-terminal tag that could be used for detection of plasmid-encoded YrpL15A is detrimental to protein function. YrpL15A could also be replaced by the homologous protein from Arabidopsis thaliana despite almost 30% differences in the amino acid sequence, while the more closely related protein from Schizosaccharomyces pombe was inactive. The lack of function was not caused by a failure of the protein to enter the yeast nucleus.

Keyword
Ribosomal protein L15, Functional complementation, Mutagenesis. Nuclear localization, Promoter, Yeast
National Category
Cell Biology
Research subject
Cellbiology
Identifiers
urn:nbn:se:su:diva-27720 (URN)10.1007/s00294-009-0228-z (DOI)000265092000002 ()
Available from: 2009-05-14 Created: 2009-05-14 Last updated: 2017-12-13Bibliographically approved

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