Degenerate localization sequences: torus markers
The marker class picked up most frequently was the torus class. Out of 120 recovered lines 43, or about 1/3, were assigned to this class. The torus phenotype is characterized by lumenal localization to small organelles, many of which have a toroid, or donut-like, appearance. At the resolution of the confocal microscope, the "hole" in the torus could either be an actual hole or an exclusion of GFP from the center of a solid organelle. For more information about the torus phenotype, see the section on the torus lines.
Three lines of evidence suggest that the torus class of tags label peroxisomes:

1. Sequencing of the torus tags reveals that many torus markers have a terminal tri-peptide motif that resembles those of defined peroxisomal targeting sequences. The canonical peroxisomal targeting motif is SKL. Recently, related tripeptides, such as SRL, have also been identified as suffincient targeting sequences. SRL is found at the termini of three torus tags. A number of other torus tags end in sequences that contain S, L, or both peptides in the -2 and last position respectively.
2. One torus tag, A5, is a likely peroxisome resident. A5 is fused to GFP in its native coding frame, and is homogous to a tetrafunctional protein isolated from peroxisomes in Brassica napis.
3. Whole-mount in situ analysis of a peroxisome resident protein, catalase, shows excellent co-localization of the A5 tag and the anti-catalase antibody.
All the sequenced torus tags except for A5 are out-of-frame fusions. The large number of torus tags recovered as out of frame fusions suggests that minimal peroxisomal targeting information is degenerate. It is confered at a relatively high frequency by semi-random carboxy terminal sequences.

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