Verlag des Forschungszentrums Jülich
JUEL-4181 Studies on the peroxisomal import of isocitrate lyases in S.cerevisiae
Sonja Meyer zu Berstenhorst
Untersuchungen zum peroxisomalen Import von Isocitrat-Lyasen in Saccharomyces cerevisiae
115 S., 2005
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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