Abstract
Although human alanine:glyoxylate aminotransferase (AGT) is imported into peroxisomes by a Pex5p-dependent pathway, the properties of its C-terminal tripeptide (KKL) are unlike those of any other type 1 peroxisomal targeting sequence (PTS1). We have previously suggested that AGT might possess ancillary targeting information that enables its unusual PTS1 to work. In this study, we have attempted to locate this information and to determine whether or not it is a characteristic of all vertebrate AGTs. Using the two-hybrid system, we show that human AGT interacts with human Pex5p in mammalian cells, but not yeast cells. Using (immuno)fluorescence microscopic analysis of the distribution of various constructs expressed in COS cells, we show the following. 1) The putative ancillary peroxisomal targeting information (PTS1A) in human AGT is located entirely within the smaller C-terminal structural domain of 110 amino acids, with the sequence between Val-324 and Ile-345 being the most likely candidate region. 2) The PTS1A is present in all mammalian AGTs studied (human, rat, guinea pig, rabbit, and cat), but not amphibian AGT (Xenopus). 3) The PTS1A is necessary for peroxisomal import of human, rabbit, and cat AGTs, but not rat and guinea pig AGTs. We speculate that the internal PTS1A of human AGT works in concert with the C-terminal PTS1 by interacting with Pex5p indirectly with the aid of a yet-to-be-identified mammal-specific adaptor molecule. This interaction might reshape the tetratricopeptide repeat domain allosterically, enabling it to accept KKL as a functional PTS1.
Highlights
To test whether this extra information is unique to human AGT or whether it is a common feature of mammalian AGTs, we have examined whether KKL is able to direct the peroxisomal import of other mammalian AGTs by substituting it for the naturally occurring C-terminal tripeptides in rat, guinea pig, rabbit, and cat AGTs
Cell Context Dependence of the Interaction between Human AGT and Human Pex5p—The fact that human AGT is able to interact with human Pex5p in mammalian cells, but is unable to do so in yeast cells (8), suggests that, unlike for other PTS1 proteins, the correct cellular environment is important for AGT
The role of this putative adaptor could be to allosterically modify the tetratricopeptide repeat (TPR) domain of Pex5p allowing it to accept the degenerate range of C-terminal tripeptides known to be able to direct human AGT to peroxisomes
Summary
KKL is necessary for peroxisomal targeting of human AGT but insufficient to direct the peroxisomal targeting of reporter proteins, such as bacterial chloramphenicol acetyltransferase or firefly luciferase (7, 12) This has led to the suggestion that additional targeting information may be necessary beyond that provided by the C-terminal tripeptide (7). Unlike the C termini of most other PTS1 proteins, the C termini of mammalian AGTs are poorly conserved It is KKL in human (9) and marmoset (20), HRL in guinea pig (21), SQL in rabbit (20), NKL in rat (22), mouse (23), and cat (24), HKL in dog (EMBL accession number AAEX01037931/2, Canis familiaris chromosome 25 contig 37930/1), and SKL in cow (EMBL accession number AV605720, Bos taurus cDNA). This evolutionary variability differs from archetypal PTS1 proteins, such as urate oxidase, the C terminus of which (i.e. SRL) is conserved in rat, rabbit, mouse, pig, and baboon, or acyl-CoA oxidase, the C terminus of which (i.e. SKL) is conserved in rat, mouse, guinea pig, human, and koala
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