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Locating the nuclear localization signal in S. cerevisiae ribosomal protein L15A
Södertörn University, School of Life Sciences.
Södertörn University, School of Life Sciences.
(English)Manuscript (preprint) (Other academic)
Resource type
Text
Abstract [en]

Newly synthesized ribosomal proteins (r-proteins) are efficiently transported from the cytoplasm to the site of ribosome assembly in the nucleolus. Nuclear import is facilitated by the presence of a nuclear localization signal (NLS) whereas the nucleolar accumulation requires a nucleolar localization signal (NoLS). In this study we located the NLS of the essential r-protein YrpL15A by studying the ability of various YrpL15A-gfp constructs to enter the nucleus and nucleolus. We found that the NLS signal is located in the C-terminal part of the protein. The identified sequence was sufficient to direct the reporter construct to the nucleus in yeast cells. This protein fragment contains a sequence that resembles a classical monopartite NLS. The fragment also contains a NoLS as seen by the partial co-localization of reporter construct with the nucleolar marker protein nop1. Orthologs of YrpL15A such as rpL15B from Arabidopsis thaliana and rpL15A from Schizosaccharomyces pombe were also able to enter the nucleus and nucleolus of yeast cells, suggesting that their NLS and NoLS are similar to that found in YrpL15. These results are discussed in relation to sequence similarities/dissimilarities. YrpL15A containing a C-terminal tag was unable to assemble into large ribosomal subunits that were transported to the cytoplasm.

Keyword [en]
Ribosomal protein L15, Functional complementation, Nuclear localization, Yeast
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:sh:diva-30699OAI: oai:DiVA.org:sh-30699DiVA: diva2:951037
Note

Som manuskript i avhandling. As manuscript in dissertation.

Available from: 2009-05-14 Created: 2016-08-05 Last updated: 2016-08-05Bibliographically approved
In thesis
1. Ribosomal proteins L5 and L15: Functional characterisation of important features, in vivo
Open this publication in new window or tab >>Ribosomal proteins L5 and L15: Functional characterisation of important features, in vivo
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein synthesis is a highly regulated and energy consuming process, during which a large ribonucleoprotein particle called the ribosome, synthesizes new proteins. The eukaryotic ribosome consists of two unequal subunits called: small and large subunits. Both subunits are composed of ribosomal RNA (rRNA) and ribosomal proteins (r-proteins).

Although rRNAs build the matrix of the ribosome and carries out catalysing of the peptide-bond formation between amino acids, r-proteins also appear to play important structural and functional roles. The primary role of r-proteins is to initiate the correct tertiary fold of rRNA and to organize the overall structure of the ribosome.

In this thesis, I focus on two proteins from the large subunit of the eukaryotic ribosome: r-proteins L5 and L15 from bakers yeast S. cerevisiae. Both r-proteins are essential for ribosome function. Their life cycle is primarily associated with rRNA interactions. As a consequence, the proteins show high sequence homology across the species borders. Furthermore, both L5 and L15 are connected to human diseases, which makes the study their role in ribosome biogenesis and ribosome function important.

By applying random- and site-directed mutagenesis, coupled with functional complementation tests, I aimed to elucidate functionally regions in both proteins, implicated in transport to the cell nucleus, protein-protein interactions and/or rRNA binding. The importance of individual and multiple amino acid exchanges in the primary sequence of rpL5 and rpL15 were studied in vivo. The obtained results show that S. cerevisiae rpL15 was tolerant to amino acid exchanges in the primary sequence, whereas rpL5 was not. Consequently, A. thaliana rpL15 could substitute for the function of wild type rpL15, whereas none of the tested orthologous proteins to rpL5 could substitute yeast rpL5 in vivo. These observations further emphasize the importance of studying r-proteins as separate entities in the ribosome context.

Place, publisher, year, edition, pages
Stockholm: Stockholm University, 2009. 50 p.
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:sh:diva-30701 (URN)978-91-7155-896-1 (ISBN)
Public defence
2009-06-16, MB 503, Södertörns högskola, Alfred Nobels allé 7, 10:00 (English)
Opponent
Supervisors
Available from: 2016-08-05 Created: 2016-08-05 Last updated: 2016-08-05Bibliographically approved

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