3. Results
3.3. Peroxisomal proteins identified by mass spectrometry
3.3.4 Evidence for peroxisomal localization of novel proteins
In order to validate the localization of the novel proteins identified from spinach leaf peroxisomes, a method based on differential profiling of proteins between a peroxisome enriched sample and a sample enriched for proplastid-like organelles was used.
3.3.4.1. Differential profiling
The major contaminant of the peroxisome fractions was the proplastid-like organelle. Therefore, fractions enriched for peroxisomes and proplastid-like organelles were taken from the same density gradient and the pattern of their constituent proteins
IEF
was resolved by two-dimensional electrophoresis. These fractions differed by a factor of more than 25 in their purity based on the ratio of HPR to NADP-GAPDH. The relative intensities of proteins known to be present in these organelles were different in these fractions that allowed distinguishing proteins that may be unique to one of them. The approach appeared specific and sensitive enough as indicated by a barely visible silver stained spot such as that of a homolog of enoyl CoA hydratase (spot no. 103, Fig. 3.11 b and arrowhead 1, Fig. 3.18) to be a peroxisomal protein. Thus the constraint with purity was overcome by a fairly simple method to deduce peroxisomal proteins.
Fig. 3.18: Subtractive two-dimensional electrophoresis to deduce peroxisomal proteins by differential profiling
Two fractions designated as peroxisomes (a) and proplastid-like organelles (b) were chosen to generate differential 2-D protein profiles with equal amounts of protein. The fractions differed in their HPR/NADP-GAPDH ratio by a factor of 25. A protein spot that was more dominant on one gel as compared to the other was considered to be located in the organelle which was enriched in that sample. The relative abundance of some of the peroxisomal proteins (CAT, GGT, and HPR) and the plastid protein RubisCO are shown. Novel proteins identified are indicated by arrowheads. 1:
enoyl CoA hydratase, 2: short chain dehydrogenase/reductase family protein, 3: naphthoate synthase like protein.
fraction enriched in peroxisomes fraction enriched in proplastid-like organelles
a b
SDS-PAGE
Mr
In summary for the novel proteins, except for the small heat shock protein, evidence could be provided in the form of differential profiling that support their peroxisomal localization. Such a differential profiling was not carried out with samples from Arabidopsis due to insufficient amounts of protein derived from most single gradients.
Instead, spots were selected based on analogy of their migration coordinates with peroxisomal proteins on spinach gels. This strategy was successful and resulted in the identification of some well-known peroxisome proteins as well as some novel ones among the selected spots.
3.3.4.2. Cloning of genes encoding novel peroxisomal proteins
Four novel proteins were detected on 2-D gels of leaf peroxisomes from spinach and Arabidopsis. Several lines of evidence strongly supported their localization in plant peroxisomes and indicated an important yet unknown function in metabolism of mesophyll cells. Because a functional hypothesis was concluded by in silico homology analyses for naphthoate synthase and because one small heat-shock protein had already been demonstrated to be targeted to plant peroxisomes (Ma and Reumann, unpublished), a subsequent goal of this thesis was cloning of the Arabidopsis genes of the naphthoate synthase homolog (At1g60550) and of the second small heat-shock protein (At1g06460).
Gene cloning is a prerequisite to verify the predicted subcellular localization of the proteins in peroxisomes by analyzing subcellular targeting of fusion proteins with spectral variants of green fluorescent protein in onion epidermal cells by fluorescence microscopy (Fulda et al., 2002; Ma, Mayer and Reumann, unpublished) as well as for functional studies of heterologously expressed recombinant proteins.
Oligonucleotide primers were designed based on the sequence of the Arabidopsis gene that is publicly available at the National Center for Biotechnological Information (NCBI) for cloning of the full-length open reading frame and supplemented with appropriate restriction sites of DNA endonucleases to facilitate subcloning. Initial attempts to amplify both full-size cDNAs gene using first strand cDNAs synthesized from total RNA of leaves failed for unknown reasons. An alternative strategy was subsequently chosen, which was based on in silico and proteome data. First, an analysis of various Arabidopsis collections of expressed sequence tags (ESTs) suggested that both genes are induced by stress
treatment, such as cold and dehydration (data not shown). Second, the proteome studies indicated that the protein spot of the small heat-shock protein increased with prolonged storage of the Arabidopsis plants in the cold prior to the isolation of leaf peroxisomes (data not shown). Thus, total RNA was subsequently isolated from Arabidopsis plants subjected to cold treatment for one hour. Indeed, full-length PCR fragments of expected size could be amplified from the first-strand cDNA derived from RNA isolated from cold-treated Arabidopsis leaves for both genes. The amplified fragments were ligated into pGEMT-easy vector. The nucleotide sequences verified cloning of the intended genes and did not reveal any exchanges at the amino acid level as compared to the protein sequence predicted from the Arabidopsis genome (AGI 2000), allowing straightforward subcloning of the genes into the E. coli expression vector pDEST17 for functional analyses.
For verification of subcellular targeting, subcloning of both full-length genes as well as of deletion constructs lacking the predicted PTS into the vector pCAT-YFP was started.
For naphthoate synthase with a predicted N-terminal PTS2, the gene is being cloning via NcoI in front of yellow fluorescent protein (YFP) to allow exposition of the PTS2 in the fusion protein, recognition by the cytosolic receptor Pex7 and targeting to peroxisomes. By contrast, the small heat-shock protein, which carries both, a predicted PTS1 (PKL>) and a PTS2 (RLx5HF), was first cloned via NotI/XbaI in the back of YFP to guarantee exposure of the C-terminal PTS1 for targeting to peroxisomes. Because subcloning of the genes into the pCAT-YFP is not trivial due to a lack of blue-white screening, the results could not be accomplished in the last weeks of experimental work of this thesis and were handed over to colleagues in the laboratory due to time constraints.