Table I.

Homology and targeting analysis of Arabidopsis proteins with a major PTS

Major PTS	Proteins	Number of Proteins		Overall Probability of Targeting to Peroxisomes
		Total		High	Medium	Low
			%	%	%	%
Total	Total	162	100	53	39	8
	Known peroxisome proteins	24	15	58	42	0
	Unknown nonhypothetical proteins	103	64	49	40	12
	Hypothetical proteins	35	22	63	34	3
PTS1	Total	131	100	66	30	5
	Known peroxisome proteins	16	12	88	12	0
	Unknown nonhypothetical proteins	90	69	56	38	7
	Hypothetical proteins	25	19	88	12	0
PTS2	Total	31	100	n.d.	77	23
	Known peroxisome proteins	8	26	n.d.	100	0
	Unknown nonhypothetical proteins	13	42	n.d.	54	46
	Hypothetical proteins	10	32	n.d.	90	10

Putative peroxisomal matrix proteins were extracted from the Arabidopsis genome based on the presence of one of nine major PTS1 tripeptides ([SA][RK][LM]> without AKM> plus SRI> and PRL>) or one of two major PTS2 nonapeptides (R[LI]x₅HL) according to a previous definition (Reumann, 2004). These proteins were classified based on homology and in silico expression. Known peroxisomal proteins (24 in total, 15%) comprise orthologs, the genes of which have been cloned from Arabidopsis or a different plant species and for which targeting of one ortholog to plant peroxisomes has been demonstrated experimentally. Unknown nonhypothetical proteins (103, 64%) mostly shared sequence similarity with eukaryotic or prokaryotic proteins, and their gene expression is supported by ESTs. In contrast to 22 of these unknown proteins annotated expressed proteins, the remaining 81 unknown nonhypothetical proteins (79%) were partly annotated and shared some sequence similarity with known proteins in the public databases. Expression of hypothetical proteins (35 proteins, 22% of all proteins with major PTSs) was not yet supported by ESTs, and some of these genes may represent pseudogenes. Targeting prediction was considered significant if two independent programs concluded subcellular targeting to the same subcellular compartment. High overall probability for targeting to peroxisomes was defined if targeting to peroxisomes was predicted with high probability at least by one program and if no nonperoxisomal targeting signal was predicted with high accuracy by two independent programs. Medium and low overall targeting probability was deduced if one or two of these criteria were not fulfilled, respectively. Due to a lack of prediction software for PTS2-targeted proteins, estimation of targeting probability was performed similarly for PTS2 proteins but restricted to the categories of medium and low probability. Further support for peroxisome targeting is provided by analysis of the targeting domain for conserved properties of PTS-targeted plant peroxisomal proteins and, for specific proteins, by analysis of PTS conservation in homologous EST (see AraPerox, Supplemental Figs. 1 and 4) but not included in the definition of overall targeting probability. n.d., Not defined.