Edited by: Peter Kijun Kim, Hospital for Sick Children, Canada
Reviewed by: Richard Rachubinski, University of Alberta, Canada; Matthias Wilmanns, European Molecular Biology Laboratory, Germany
This article was submitted to Membrane Traffic, a section of the journal Frontiers in Cell and Developmental Biology
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Import of peroxisomal matrix proteins with a type 1 peroxisomal targeting signal (PTS1) in
Peroxisomes are ubiquitous cell organelles with variable content of enzymes depending on cell types and environmental or developmental conditions. Peroxisomal enzymes are posttranslationally imported as fully folded and oligomerized proteins and assisted by cytosolic receptor proteins. Most peroxisomal matrix proteins harbor a C-terminal peroxisomal targeting signal type 1 (PTS1), which is recognized by the PTS1-receptor Pex5p (
Upon interaction between the cytosolic receptor-cargo complexes with the docking complex at the peroxisomal membrane, Pex5p or Pex18p associate Pex14p to assemble the respective protein conducting channels of the PTS1- and PTS2-pathway (
The set of membrane bound peroxins enabling the cycling of import receptors is well conserved between yeast and other organisms. However, yeast cells use differentially expressed receptors, like the PTS2 co-receptors Pex18p and Pex21p (
Here, we mapped the Pex9p-mediated protein import pathway by analyzing Pex9p-dependent protein import in various
The
Cells transformed with GFP fused to PTS1 signal of Pcs60p (pLDC14) or Mls1p (pDE07), with additional Pex9p-ProtA (pMAR28) or Pex9C6A-ProtA (pMAR29) for complementation studies, were grown in YNBGO medium for 16 h. Formaldehyde was added to a final concentration of 3.7% and the culture was incubated for 10 min at RT with gentle agitation. After sedimentation (13.000 rpm, 1 min), cells were resuspended in 0.1M KH2PO4 (pH 6.8) supplemented with 3.7% formaldehyde. The cells were incubated for 1 h at RT with gentle agitation, sedimented, resuspended in 0.1M KH2PO4 (pH 6.8), containing 1% ethanolamine and incubated for further 10 min as indicated above. Afterward, cells were washed twice with PBS (pH 7.5). Finally, the cells were resuspended in a small volume of PBS, subjected to slides, and sealed with a cover slip.
For wide-field fluorescence imaging, a Zeiss Axioskop 50 fluorescence microscope (Zeiss) was used. A Princeton Instruments 1300Y digital camera was used for image acquisition. GFP fluorescence was visualized using a 450–490 nm band pass excitation filter, a 510 nm dichromatic mirror, and a 515–565 nm band pass emission filter (
Samples were taken from cells grown in YNGBO medium for 12 h (0 h) before either cycloheximide (final concentration 0.1 mg/ml, dissolved in DMSO) or only DMSO was added to the cultures. Cultures were grown at 30°C and samples were taken at the indicated time points. To generate total protein samples, a modified protocol from
Cells deficient in both Pex5p and Pex9p transformed with GFP-Mls1p in addition to Pex9p fused to ProteinA under its endogenous promoter (pMAR28) or a variant in which the conserved cysteine (Cys6) was substituted to alanine (pMAR29) were grown in YNBGO medium for 12 h. Of these, total protein samples were generated as described above.
Pex9p was shown to associate with the peroxisomal membrane under oleate-inducing conditions (
Dependence of Pex9p-related protein import on known components of the peroxisomal protein import machinery. Wild-type (WT) cells and yeast mutants deleted in one or more PEX genes (Δ
To compare the life-span of Pex9p and Pex5p, the strains UTL7A Pex9p-ProteinA expressing genomically tagged Pex9p under the control of its own promoter (
Rapid turnover rate of Pex9p under oleate-inducing conditions. Yeast UTL7A cells expressing ProteinA-tagged Pex9p
Even though we found that the same import machinery is required for both PTS1 receptors, it was not yet clear whether Pex9p really undergoes a cyclic processing as Pex5p does. A checkpoint in Pex5p recycling is monoubiquitination of a unique cysteine close to the N-terminus (
Identification of Pex9p from the pathogenic yeast
Cys6 of Pex9p is critical for protein import into peroxisomes.
The import cycle of the import receptor Pex5p requires the sequential action of several peroxins that contribute to receptor docking, pore formation, ubiquitination and ATP-dependent export and recycling. Here, we showed that the novel PTS1-receptor Pex9p requires the same set of membrane-associated peroxins to facilitate protein import into peroxisomes as Pex5p and PTS2 receptors. In analogy to the Pex5p-dependent protein import into peroxisomes, a complete picture of the Pex9p-dependent import into peroxisomes can now be postulated. According to this model, the Pex9p-cargo complex docks at a membrane complex consisting of Pex13p, Pex14p and Pex17p. High binding affinity between Pex9p and Pex14p was shown previously, while the other two proteins of this complex displayed no visible interaction (
Although the import pathways of Pex5p and Pex9p utilize the same components, the fate of the extracted PTS1 receptors is different. Where Pex5p is mostly recycled to enable further rounds of import, the bulk of Pex9p is rapidly degraded under oleate-induced conditions. This is underlined by different turnover rates. While the half-life of constitutively expressed Pex5p was estimated to be 2.5 h on glucose medium (
Interestingly, the turnover rate of biologically inactive Pex9C6A in oleate grown cells is the same as for wild type. This suggests that Pex9p degradation occurs independently from functional import. The mechanisms underlying the fast degradation process of Pex9p are not known. However, other peroxisomal receptor variants which are impaired in monoubiquitination and thereby, get stuck in the import pathway are degraded by proteasomes in a kind of quality control (
The rapid turnover rate of Pex9p correlates with transcriptional regulation and function of the PTS1- receptor. Pex9p is expressed under specific growth conditions and highly selective for a limited number of PTS1 cargo proteins. The known cargo enzymes, malate synthases Mls1p and Mls2p, are catalytically fully active in the cytosol and required for metabolism in cells grown in glucose, ethanol or acetate. Upon shift on oleate medium, the enzymes are transported into peroxisomes where fatty acids are oxidized to metabolites which are further converted by malate synthases. In other words, the activity of Pex9p helps to optimize yeast metabolism during growth on fatty acid as carbon source. The short lifetime of Pex9p combined with a carbon source-dependent transcriptional regulation enables the yeast to adapt the intracellular localization of malate synthases as a fast response to environmental changes, like a shift from oleate to other carbon sources. The efficacy of a “switchable” Pex9p-dependent import pathway is ensured since this route relies on an already existing peroxin network at the peroxisomal membrane. By sequence comparison, we identified a putative ortholog of the alternative PTS1-receptor Pex9p in a different yeast species, the pathogenic fungus
The differences in the regulation of Pex5p and Pex9p abundance show parallels to PTS2 import, which engages two paralogous co-receptors, Pex18p and Pex21p. These proteins were identified and characterized in
All datasets presented in this study are included in the article/
MR, RE, and WS conceived and planned the experiments, contributed to the interpretation of the results, and writing of the manuscript. MR carried out the experiments and analyzed the data. WS performed the sequence analyses. All authors contributed to the article and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Luis Daniel Cruz-Zaragoza for fruitful discussions and advice. We also thank all former lab members contributing to the generation of the yeast strains utilized in this study. We acknowledge support by the DFG Open Access Publication Funds of the Ruhr-Universität Bochum.
The Supplementary Material for this article can be found online at: