Funktionelle Analyse des Hypusin enthaltenden Proteins in Saccharomyces cerevisiae

Hypusine is an unusual and unique posttranslational modification,
conserved in evolution from halobacteria to man. However, despite
being essential for life the function of the modification and the
protein carrying it (Hypp) remains unclear so far. In yeast
temperature sensitive mutants were generated and phenotypicly
characterized. The investigation included proteomic and
transcriptomic techniques. Furthermore binding assays using several
Hypp fusion proteins were performed in order to find cellular
protein and RNA interaction partners. Describing the phenotype of a
highly temperature sensitive point mutated allele of Hypp a limited
viability at restrictive temperature was shown leading to apoptotic
cell death suggesting an antiapototic property of the protein. On
the transcriptomic level studies using high density oligonucleotide
micro arrays covering the whole yeast genome were performed. As
also found on the protein level by 2D-gel analysis the impairment
of mitochondrial functions was confirmed. Respectively 70% of the
enriched transcripts of the mutant were also accumulated in strains
in which components of the NMD pathway were disrupted indicating a
function of the hypusine containing protein in this special
5’-3’-mRNA degradation pathway to which NMD belongs. A respiratory
deficiency also was the first observed phenotype of strains bearing
a NMD dysfunction. The mutant showed an attenuation of the telomer
positioning effect leading to elevated transcriptional activity of
genes near the telomers. It could be demonstrated that the Hypp
mutant showed shortening of telomers similarly observed in NMD
deficient strains. No evidence for the protein to be a general
nuclear export factor of a special RNA subset could be given. By
affinity chromatographic purification of N-terminal GST-Hypp fusion
proteins an interaction of Hypp to the major coat protein of the
yeast specific virus L-A was demonstrated, a protein that itself
posesses an enzymatic activity for the decapping of mature RNA
molecules. Before it was shown that N-terminally GST-tagged Hypp
was able to take over the function of the wild-type protein
unrestrictedly. Our results give evidence that the protein posesses
cellular cytosolic functions in the posttranscriptional control of
gene activity of viral and normal genes in Saccharomyces
cerevisiae. A connection to a special RNA degradation pathway is
evident and should be investigated further on.