Verlag des Forschungszentrums Jülich

JUEL-4181
Sonja Meyer zu Berstenhorst
Untersuchungen zum peroxisomalen Import von Isocitrat-Lyasen in Saccharomyces cerevisiae
115 S., 2005



Studies on the peroxisomal import of isocitrate lyases in S.cerevisiae

Peroxisomes play a central role in the metabolism of fatty acids and the detoxication of reactive oxygen species, e.g. H2O2 , emerging from oxidation processes. Peroxisomal disorders cause diseases, often due to an import defect of certain enzymes. The two major import pathways of peroxisomal matrix proteins are based on conserved signal sequences. The most common signal is the so-called PTS1, a tripeptide at the end of the C-terminus. More rarely an N-terminal nonapeptide (PTS2) occurs. Moreover some proteins contain an additional signal and/or use a hitherto unknown import pathway. For instance only some peroxisomal isocitrate lyases have a PTS1 signal.
Isocitrate lyase (ICL) is a key enzyme of the glyoxylate pathway and thus essential for growth on non-fermentable carbon sources. Eukaryotic ICLs are usually localized in the peroxisomes, with the exception of the cytosolic ICL of the baker’s yeast S.cerevisiae. In contrast to this cytosolic Sc ICL the peroxisomal Ashbya gossypii -ICL contains a PTS1 signal. Therefore the question arose, whether this signal was sufficient to mediate the peroxisomal ICL-import or whether an additional signal was necessary.
By fusion of the PTS1-tripeptide AKL or exchange of the C-terminus with that of Ag ICL an import of 3 - 4% of the modified Sc ICL was achieved. In the course of this the peroxisomal localization was independently shown by sucrose density centrifugation as well as immuno electron microscopy. Interestingly, the import of heterologously expressed Ag ICL was far more efficient with approx. 20%. This difference pointed to an additional peroxisomal signal of the Ag ICL compared with the Sc ICL. The thesis was supported by muteins without a functional PTS1, which were imported to 20% ( Ag ICL extended by 39 amino acids) or 9% (PTS1 deleted). The signal needed for this import was assumed within the region of the structural domain II of the Ag ICL and researched with a hybrid protein of the E.coli -ICL. The ICL structural domain II is an internal region of about 100 amino acids, which is missing in prokaryotes. Thus the hybrid protein Ec ICL Ag DII was made by insertion of the Ag ICL structural domain II ( Ag DII). Differential centrifugations displayed a significant peroxisomal localization of this hybrid protein. While - according to activity assays - only 0.7% of the wild type Ec ICL located in the yeast organelles, the proportion of the hybrid protein was 33%. The result was confirmed by Western blots presenting a clear band of Ec ICL Ag DII within the organellar pellet. Thereby the functionality of an ICL structural domain II sequence as an organellar signal was shown for the first time.
In castor bean ICL the PTS1 region is also dispensable for peroxisomal import, which nevertheless requires the PTS1 receptor PEX5 (Parkes et al, 2003). To check whether this relationship also exists for the PTS1-less Ag ICL and the hybrid Ec ICL Ag DII, a PEX5 disruption mutant was cloned. The unaltered subcellular distribution of both ICL muteins in this strain background contradicted a dependency of the import on PEX5. Enzyme assays demonstrated 8% of the PTS1-less Ag ICL or 34% of the hybrid Ec ICL Ag DII within the organellar pellet. The localization of the ICLs within the organelles as well as the mislocalization of the PEX5- dependent catalase A were confirmed by Western blots. Immuno-detection within the fractions of sucrose density gradients showed the co-localization of both ICL muteins with thiolase, a PTS2 peroxisomal marker. Hence the Ag ICL contained a novel, PEX5-independent peroxisomal signal within region of its structural domain II.

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Letzte Änderung: 07.06.2022