Journal: bioRxiv

Article Title: PEX39 facilitates the peroxisomal import of PTS2 proteins

doi: 10.1101/2024.04.30.591961

Figure Lengend Snippet: (A) Loss of Sc Pex39 confers a fitness defect in yeast grown in oleic acid. Cells were precultured in 0.3% glucose medium for 16 h at 30°C and then shifted to oleic-acid medium for further growth. For complementation, Scpex39Δ cells were transformed with a plasmid containing Scpex39 under control of its endogenous promoter (pPEX39). Cell growth was monitored by measuring the OD600 at the indicated time points. Data are mean ± standard deviation (SD) (n = 4). Error bars may not be visible if the SD is very small. (B) Loss of Sc Pex39 specifically impairs PTS2-protein import in yeast per cellular fractionation. A post-nuclear supernatant (PNS) was prepared from oleic acid-grown wild-type and Scpex39Δ cells and was further separated into a cytosolic fraction (supernatant, S) and an organellar pellet (OP). Equal volumes of the fractions were analyzed by immunoblotting for the indicated proteins. The experiment was performed in three independent replicates (see also Figure S2B). (C) Quantification of changes in the subcellular distribution of PTS2 proteins upon loss of Sc Pex39. Signal intensities of immunoblots shown in (B) and Figure S2B were quantified using ImageJ. For each protein, intensities for the cytosolic supernatant (S) and the organellar pellet (OP) were normalized to the PNS and the sum was set to 100%. Data are mean ± standard error of the mean (SEM) (n = 3), and P values were calculated using unpaired, two-tailed t-tests. (D) Loss of Sc Pex39 impairs import of Pot1 in yeast per fluorescent microscopy. Localization of Pot1-mNeonGreen (C-terminal tagging) was examined in control, Scpex39Δ , and pex7Δ cells after 8 h of growth on oleic acid. Peroxisomes were visualized with Pex3-mScarlet. Scale bar: 1 μm. (E) Loss of Sc Pex39 results in decreased levels of Pex7. Whole cell lysates of wild-type (WT) and Scpex39Δ cells were analyzed by SILAC-based quantitative mass spectrometry (n = 4 biological replicates). Shown are proteins quantified in at least three biological replicates, except for Sc Pex39, which was quantified in only one replicate. PTS2 proteins are highlighted in yellow; further peroxisomal proteins are marked by black dots. Adjusted P values were determined using the “linear models for microarray data” (limma) approach. Dashed horizontal line indicates an adjusted P value threshold of 0.05. (F) Precursor and mature PHYH increase and decrease, respectively, in human HsPEX39 -knockout cells. CRISPR-Cas9 was used to generate knockouts ( HsPEX39 -KO) and matched controls (Control) in the CAKI-2 and NCI-H1792 cell lines (see STAR Methods). Cellular lysates were analyzed by immunoblotting for the indicated proteins. For the PHYH, ACAA1, and AGPS blots, the solid and open red arrowheads indicate the mature and precursor forms of these proteins, respectively. Precursor forms of ACAA1 and AGPS were undetectable in these experiments. CANX and CS are loading controls. Short and long exposures are denoted s.e. and l.e., respectively. (G) Quantification of changes in mature and precursor PHYH in HsPEX39 -knockout cells. Band intensities of immunoblots prepared per (F) were quantified using ImageJ. Data are mean ± SEM (n = 3), and P values were calculated using unpaired, two-tailed t-tests. See also Figure S2.

Article Snippet: Cellular fractionation was performed using the Cell Fractionation Kit (Cell Signaling Technology 9038) generally according to the manufacturer’s instructions.

Techniques: Transformation Assay, Plasmid Preparation, Control, Standard Deviation, Cell Fractionation, Western Blot, Two Tailed Test, Microscopy, Multiplex sample analysis, Mass Spectrometry, Microarray, Knock-Out, CRISPR

Journal: bioRxiv

Article Title: PEX39 facilitates the peroxisomal import of PTS2 proteins

doi: 10.1101/2024.04.30.591961

Figure Lengend Snippet: (A) Overexpression of Hs PEX39 increases precursor forms and decreases mature forms of PHYH, ACAA1, and AGPS in human cells. GAPDH (negative control) or Hs PEX39 were stably overexpressed in HEK293T cells and cellular lysates analyzed by immunoblotting for the indicated proteins. For the PHYH, ACAA1, and AGPS blots, the solid and open red arrowheads indicate the mature and precursor forms of these proteins, respectively. CANX is a loading control. (B) Quantification of changes in mature and precursor forms of PHYH, ACAA1, and AGPS upon Hs PEX39 overexpression. Band intensities of immunoblots prepared per were quantified using ImageJ. Data are mean ± SEM (n = 3), and P values were calculated using unpaired, two-tailed t-tests. (C) Exogenously added Hs PEX39 inhibits peroxisomal import of ACAA1 in vitro . 35 S-ACAA1 in vitro import assays at 37°C in the presence of increasing concentrations of H6 Hs PEX39 as indicated. After incubation, reactions were treated with trypsin and organelles were isolated by centrifugation and analyzed by SDS-PAGE and autoradiography; protection from trypsin and maturation of ACAA1 reflects import into peroxisomes. Precursor and mature forms of ACAA1 denoted by open and solid red arrowheads, respectively. Numbers along the left side of each image indicate molecular weights (kD). See STAR Methods for detailed description of this assay. (D) Overexpression of Sc Pex39 confers a fitness defect on yeast grown on oleic acid. Experiment performed as described in . Scpex39Δ cells were transformed with a plasmid containing Scpex39 under control of a TEF2 promoter (pPEX39_OE) for overexpression. Data for wild-type, Scpex39Δ and Scpex39Δ + pPEX39 are the same as shown in (right plot). Data are mean ± SD (n = 4). Error bars may not be visible if the SD is very small. (E) Cellular fractionation of yeast overexpressing Sc Pex39. Experiment performed as described in using Scpex39Δ cells transformed with plasmid pPEX39 (endogenous promoter) or pPEX39_OE (TEF2 promoter) for Sc Pex39 overexpression. PNS, post-nuclear supernatant; S, cytosolic supernatant; OP, organellar pellet. See also Figure S3.

Article Snippet: Cellular fractionation was performed using the Cell Fractionation Kit (Cell Signaling Technology 9038) generally according to the manufacturer’s instructions.

Techniques: Over Expression, Negative Control, Stable Transfection, Western Blot, Control, Two Tailed Test, In Vitro, Incubation, Isolation, Centrifugation, SDS Page, Autoradiography, Transformation Assay, Plasmid Preparation, Cell Fractionation

Journal: bioRxiv

Article Title: PEX39 facilitates the peroxisomal import of PTS2 proteins

doi: 10.1101/2024.04.30.591961

Figure Lengend Snippet: (A) Investigation of Hs PEX39 truncation and mutated variants using native-PAGE and radiolabeled PEX7. Depictions of the different variants are shown on the left. 35 S-H 6 PEX7 was incubated or not with the indicated recombinant proteins and analyzed by native-PAGE and autoradiography. In-gel position of PEX7 alone (PEX7), lysate hemoglobin, and of the complexes PEX7- Hs PEX39 (#), PEX7- Hs PEX39-PHYH (&), PEX7-PEX5-PHYH- Hs PEX39 ($) and PEX7-PEX5-PHYH (*) are indicated for the full-length wild-type Hs PEX39 variant. Double bands in Hs PEX39(ΔN) complexes are due to co-migration with hemoglobin from the lysate. (B) Investigation of Hs PEX39 truncation and mutated variants using an in vitro import assay. 35 S-ACAA1 was subjected to in vitro import assays at 37°C in the absence (-) or presence of the indicated recombinant Hs PEX39 proteins [see depictions of the indicated variants in left side of (A)]. After incubation, reactions were treated with trypsin and organelles were isolated by centrifugation and analyzed by SDS-PAGE and autoradiography. Precursor and mature forms of ACAA1 denoted by open and solid red arrowheads, respectively. I, 5% of the reticulocyte lysate containing the 35 S-labeled protein used in each reaction. (C) Mutation of the RPWE motif in Sc Pex39 prevents rescue of the fitness defect of Scpex39Δ cells grown on oleic acid. Experiment performed as described in analyzing the growth of Scpex39Δ cells transformed with a plasmid for expression of an Sc Pex39 RPWE-to-AAAA mutant under control of the endogenous promoter [pPEX39(4A)] in oleic acid medium. Data for wild-type, Scpex39Δ , and Scpex39Δ + pPEX39 are the same as shown in (right plot). Data are mean ± SD (n = 4). Error bars may not be visible if the SD is very small. (D) Cellular fractionation of Scpex39Δ yeast expressing wild-type or mutant Sc Pex39. Experiment performed as described in using Scpex39 Δ cells transformed with plasmids for expression of wild-type Sc Pex39 (pPEX39) or the Sc Pex39 RPWE-to-AAAA mutant [pPEX39(4A)], each under control of the endogenous promoter. PNS, post-nuclear supernatant; S, cytosolic supernatant; OP, organellar pellet. (E) Mutations of the KPWE motif of Hs PEX39 have deleterious effects on protein interactions per assessment with HEK293T cells. Anti-FLAG immunoprecipitates and cell lysates were prepared from wild-type HEK293T cells stably expressing the indicated proteins. Samples were analyzed by immunoblotting for the indicated proteins; detection of HA denoted by “ HA ” in the labeled black arrowheads identifying the corresponding proteins. GAPDH-FLAG-HA is a negative control for the immunoprecipitations. Different Hs PEX39 variants denoted as WT (wild-type) or by single or multiple alanine replacements of the indicated residue(s). (F) Close-up views of the interactions between the KPWE motif of Hs PEX39 and PEX7. Shown are different surface properties and relative sequence conservation of the Hs PEX39 binding region at the bottom face of PEX7. Images are based on the same predicted structural model as shown in ( H. sapiens ). The individual amino acids of the KPWE motif are labeled. (G) Mutation of the KPWE motif prevents overexpressed Hs PEX39 from impairing the import of PHYH, ACAA1, and AGPS in human cells. GAPDH (negative control), Hs PEX39, or Hs PEX39 with KPWE motif replaced by AAAA [ Hs PEX39(4A)] were stably overexpressed in wild-type HEK293T cells and cellular lysates analyzed by immunoblotting for the indicated proteins. For the PHYH, ACAA1, and AGPS blots, the solid and open red arrowheads indicate the mature and precursor forms of these proteins, respectively. CANX is a loading control. See also Figure S5.

Article Snippet: Cellular fractionation was performed using the Cell Fractionation Kit (Cell Signaling Technology 9038) generally according to the manufacturer’s instructions.

Techniques: Clear Native PAGE, Incubation, Recombinant, Autoradiography, Variant Assay, Migration, In Vitro, Isolation, Centrifugation, SDS Page, Labeling, Mutagenesis, Transformation Assay, Plasmid Preparation, Expressing, Control, Cell Fractionation, Stable Transfection, Western Blot, Negative Control, Residue, Sequencing, Binding Assay

Journal: bioRxiv

Article Title: PEX39 facilitates the peroxisomal import of PTS2 proteins

doi: 10.1101/2024.04.30.591961

Figure Lengend Snippet: (A) Schematic demonstrating that PEX39 and the N-terminus of PEX13 both possess [R/K]PWE motifs in yeast and humans. Select portions of the respective amino acid sequences are shown, with the [R/K]PWE motifs highlighted in purple (PEX39) or orange (PEX13). (B) The KPWE motif is necessary for the N-terminus of Hs PEX13 to bind PEX7 in vitro . Radiolabeled H6PEX7 was pre-incubated with the recombinant proteins as indicated. NtPEX13, first 36 residues of Hs PEX13 fused to the N-terminus of the small ubiquitin-related modifier 1 (Sumo1) with a hexa-histidine tag at the C-terminal end; NtPEX13(4A), NtPEX13 with KPWE motif mutated to AAAA. Samples were analyzed by native-PAGE and autoradiography. In-gel migration of PEX7 alone (PEX7), lysate hemoglobin, and of the complexes PEX7-PEX5-PHYH (*) and PEX7- Hs PEX13 (@) are indicated. (C) Assessment of KD,app for the interaction between N-terminus of Hs PEX13 and PEX7. 35 S-labeled H6PEX7 was incubated with increasing amounts of NtPEX13 as indicated and analyzed by native-PAGE and autoradiography. In-gel migration of PEX7 (PEX7), lysate hemoglobin, and of the complex PEX7- Hs PEX13 (@) are indicated. (D) AlphaFold prediction of interactions between the PEX13 N-terminus and PEX7 in human and yeast. Top and bottom faces of PEX7 are oriented as indicated. (Left) The KPWE motifs of PEX13 are marked with a dotted circle. AlphaFold prediction was performed using full-length sequences of PEX7 and amino acids 1-55 of PEX13. For visualization, PEX13 was C-terminally shortened at residue 36 as indicated. (Right) AlphaFold confidence scores of corresponding models. pLDDT, predicted local distance difference test. (E) Mutation of the N-terminal KPWE motif in Sc Pex13 confers a fitness defect in yeast grown on oleic acid. Experiment performed as described in . Scpex13Δ cells were transformed with plasmids expressing wild-type Sc Pex13 (pPEX13) or a Pex13 mutant in which the KPWE motif was converted to AAAA [pPEX13(4A)] via the endogenous promotor. Data are mean ± SD (n = 4). Error bars may not be visible if the SD is very small. (F) Cellular fractionation of Scpex13Δ yeast expressing wild-type or mutant Sc Pex13. Experiment performed as described in using cells expressing plasmid-encoded wild-type Sc Pex13 (pPEX13) or the KPWE-to-AAAA mutant [pPEX13(4A)] described in (E). PNS, post-nuclear supernatant; S, cytosolic supernatant; OP, organellar pellet. See also Figure S6.

Article Snippet: Cellular fractionation was performed using the Cell Fractionation Kit (Cell Signaling Technology 9038) generally according to the manufacturer’s instructions.

Techniques: In Vitro, Incubation, Recombinant, Ubiquitin Proteomics, Clear Native PAGE, Autoradiography, Migration, Labeling, Residue, Mutagenesis, Transformation Assay, Expressing, Cell Fractionation, Plasmid Preparation