The Peroxisomal Targeting Signal 3 (PTS3) of the Budding Yeast Acyl-CoA Oxidase Is a Signal Patch.

Błażej Kempiński,Ralf Erdmann,Łukasz Rymer,Kamil Król,Wolfgang Girzalsky,Anna Chełstowska,Jarosław Poznański,Marek Skoneczny,Zuzanna Frydzińska,Adrianna Skoneczna

doi:10.3389/fcell.2020.00198

Abstract

The specificity of import of peroxisomal matrix proteins is dependent on the targeting signals encoded within their amino acid sequences. Two known import signals, peroxisomal targeting signal 1 (PTS1), positioned at the C-termini and PTS2 located close to N-termini of these proteins are recognized by the Pex5p and Pex7p receptors, respectively. However, in several yeast species, including Saccharomyces cerevisiae, proteins exist that are efficiently imported into peroxisomes despite having neither PTS1 nor PTS2 and for which no other import signal has been determined. An example of such a protein is S. cerevisiae acyl-CoA oxidase (AOx) encoded by the POX1 gene. While it is known that its import is driven by its interaction with the N-terminal segment of Pex5p, which is separate from its C-terminal PTS1-recognizing tetratricopeptide domain, to date, no AOx polypeptide region has been implicated as critical for this interaction, and thus would constitute the long-sought PTS3 signal. Using random mutagenesis combined with a two-hybrid screen, we identified single amino acid residues within the AOx polypeptide that are crucial for this interaction and for the peroxisomal import of this protein. Interestingly, while scattered throughout the primary sequence, these amino acids come close to each other within two domains of the folded AOx. Although the role of one or both of these regions as the PTS3 signal is not finally proven, our data indicate that the signal guiding AOx into peroxisomal matrix is not a linear sequence but a signal patch.

Highlights

The mechanisms by which the majority of proteins are imported into the peroxisomal lumen are well known and are based on the recognition of short stretches of amino acids located either at the C-terminus or near the N-terminus (PTS2) of their polypeptides by receptors that recognize these signals, Pex5p or Pex7p, respectively (Girzalsky et al, 2010)
For the search of import signal(s) that direct S. cerevisiae acyl-CoA oxidase (AOx) to the peroxisomal matrix, we used a two-hybrid screen combined with random mutagenesis of the POX1 ORF
The library of mutated versions of the POX1 ORF that were fused in frame to sequence encoding the Gal4 DNA-binding domain in the pGBT9 vector was screened with the fragment of the PEX5 gene between 136 and 292 codon fused in frame to the sequence encoding the Gal4-activating domain in the pGAD424 vector

Summary

Introduction

The mechanisms by which the majority of proteins are imported into the peroxisomal lumen are well known and are based on the recognition of short stretches of amino acids located either at the C-terminus (peroxisomal targeting signal 1, PTS1) or near the N-terminus (PTS2) of their polypeptides by receptors that recognize these signals, Pex5p or Pex7p, respectively (Girzalsky et al, 2010). The region between amino acid residues 250 and 270 with the Ile264 plays a crucial role in AOx recognition and import (Klein et al, 2002) This surprising finding portrays Pex5p as a dualdomain, dual-function peroxisomal import receptor and suggests that the mechanism of AOx import may converge with the well-established PTS1-dependent import route. We recently demonstrated that two other peroxisomal proteins, (1) the multifunctional enzyme of the peroxisomal fatty acid beta-oxidation pathway that comprises the 3-hydroxyacyl-CoA dehydrogenase and enoylCoA hydratase (Fox2p) and (2) the peroxisomal catalase A, both containing the PTS1 signals at their C-termini, are partially dependent on this side branch of the Pex5p-dependent import route (Rymer et al, 2018) It remains an open question whether the list of S. cerevisiae proteins interacting with the N-terminal half of the Pex5p en route to peroxisomes is complete. While the very existence of this side branch of the Pex5p-dependent import mechanism employed by AOx is well established, to date, we do not know the corresponding module within the AOx molecule that is recognized by the N-terminal region of the Pex5p receptor

Methods

Results

Conclusion