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. 2016 Nov 21;215(4):515-529.
doi: 10.1083/jcb.201602064. Epub 2016 Nov 8.

A Family of Membrane-Shaping Proteins at ER Subdomains Regulates Pre-Peroxisomal Vesicle Biogenesis

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Free PMC article

A Family of Membrane-Shaping Proteins at ER Subdomains Regulates Pre-Peroxisomal Vesicle Biogenesis

Amit S Joshi et al. J Cell Biol. .
Free PMC article

Abstract

Saccharomyces cerevisiae contains three conserved reticulon and reticulon-like proteins that help maintain ER structure by stabilizing high membrane curvature in ER tubules and the edges of ER sheets. A mutant lacking all three proteins has dramatically altered ER morphology. We found that ER shape is restored in this mutant when Pex30p or its homologue Pex31p is overexpressed. Pex30p can tubulate membranes both in cells and when reconstituted into proteoliposomes, indicating that Pex30p is a novel ER-shaping protein. In contrast to the reticulons, Pex30p is low abundance, and we found that it localizes to subdomains in the ER. We show that these ER subdomains are the sites where most preperoxisomal vesicles (PPVs) are generated. In addition, overproduction or deletion of Pex30p or Pex31p alters the size, shape, and number of PPVs. Our findings suggest that Pex30p and Pex31p help shape and generate regions of the ER where PPV biogenesis occurs.

Figures

Figure 1.
Figure 1.
Overexpression of Pex30p and Pex31p restores ER shape in rtn1rtn2yop1Δ cells. (A) Schematic representation of the screen for genes that complement rtn1rtn2yop1spo7Δ cells. (B) The indicated strains were grown to mid-logarithmic growth phase, serially diluted, spotted onto plates that do or do not contain 5-fluoroorotic acid (5-FOA), and incubated at 30°C for 3 d. (C) Fluorescence microscopy images of strains expressing ss-RFP-HDEL focusing on the center (top) or periphery (bottom) of the cells. Arrows indicate normal cortical ER in wild-type (WT) and abnormal cortical ER in mutant. Bars, 3 µm. (D) Quantification of the experiment in C: Percentage of cells with normal ER morphology (mean ± SE; n = 3 biological replicates of at least 200 cells). (E) EM images of indicated strains. Yellow arrows indicate cortical ER; blue arrows, plasma membrane (PM); and green arrows, cell wall (CW). Bottom panel shows ER in yellow. Bars: (wild type) 200 nm; (rtn1rtn2yop1Δ) 100 nm. N, nucleus; V, vacuole.
Figure 2.
Figure 2.
Pex30p tubulates membrane in vivo and in vitro. (A) Pex30p-Flag was purified from S. cerevisiae and analyzed by SDS-PAGE followed by staining with Coomassie blue. (B) Pex30p-Flag in LDAO was mixed with EPL, incubated with Bio-Beads for 4 h, and visualized by negative-stain EM. (C) As in B, but without EPL. (D) As in B, but without protein. Bars, 200 nm. (E) Pex30p in LDAO was subjected to sucrose density-gradient centrifugation before (top) or after incubation with EPL, a fluorescent phospholipid, and Bio-Beads to remove LDAO (bottom). Fractions were immunoblotted with an anti-Flag antibody. (F) Relative amount of fluorescent phospholipid in the fractions from the bottom panel of E (EPL + Pex30p) and an identical experiment without protein (EPL). (G) Fluorescence microscopy images of the periphery of wild-type (WT) and opi1Δ cells expressing Rtn1p-GFP (green) and ss-RFP-HDEL (red). Bar, 3 µm. (H) Quantification of the experiments in G. The relative area of ER sheets was determined from the area of ss-RFP-HDEL fluorescence that did not colocalize with Rtn1p-GFP fluorescence (mean ± SE; n = 16). M, molecular mass markers (in kD).
Figure 3.
Figure 3.
Pex30p and Pex31p are reticulon like ER-shaping proteins. (A) Fluorescence microscopy (FM) images of rtn1rtn2yop1Δ cells expressing ss-RFP-HDEL and Pex30-GFP or Pex31-GFP from high-copy plasmids. Bar, 3 µm. (B) Predicted domain architecture of Pex30p; reticulon-like domain was identified using HHpred. The DysF domain has two parts, DysFN and DysFC. (C) Conserved tryptophan shown in red in the aligned amino acid sequence of Rtn1p, Rtn2p, Pex30p, and Pex31p using HHpred. (D) Serial dilutions of cultures of the indicated strains as in Fig. 1 B. (E) FM images of cortical ER in the indicated strains expressing Sec63-GFP. Bar, 3 µm. (F) Heterozygous spo7Δpex30Δpex31Δ diploid cells were sporulated, and the haploid spores were separated and grown on plates. Genotypes of selected haploids are indicated. 5-FOA, 5-fluoroorotic acid.
Figure 4.
Figure 4.
Pex30p localizes to subdomains of the ER. (A) Fluorescence microscopy (FM) images expressing endogenous Pex30-2xmCherry and Sec63-GFP focusing on the center or periphery of cells. Yellow arrows indicate Pex30-GFP subdomains in ER tubules, and white arrows indicate Pex30-GFP at the edges of ER sheets. Bar, 3 µm. (B) Immunoblots of whole-cell extracts from the indicated strains; equal amounts of protein were separated using 4–12% SDS-PAGE. (C) FM images (top) of cells expressing endogenous Pex30-2xmCherry and Rtn1-GFP. The intensity of the pixels was measured using ImageJ. Bar, 3 µm. The line profile graph (bottom) shows the pixel intensities for Pex30-2xmCherry (red) and Rtn1-GFP (green) along the line drawn across the cell as shown in the figure. (D) Cell lysate were fractionated by centrifugation on a Histodenz step gradient. Pex30p cofractionated with the ER marker Dpm1p. Pex14-GFP was used as a marker for peroxisomes.
Figure 5.
Figure 5.
Pex30p and Pex31p play a role in PPV biogenesis. (A) Peroxisome biogenesis was induced as described in Materials and methods; graph shows percentage of cells containing mature peroxisomes at indicated time points after induction of Pex3p expression (n = 3). (B) Immunoblots of the strains in A with antibodies against Pex3p and anti-porin (as a loading control). (C) Mean number of Pex14-GFP puncta in the indicated strains; mean ± SE of three replicates, at least 100 punctae counted per replicate (*, P < 0.05; **, P < 0.005). (D) The results in C were regraphed to show the mean number of cells with the indicated number of Pex14-GFP punctae.
Figure 6.
Figure 6.
Pex14-GFP containing PPVs originate in the ER. (A) Fluorescence microscopy (FM) images of pex3atg1Δ cells expressing endogenously tagged Pex14-GFP and Sec63-mCherry expressed from a plasmid. Arrows indicate Pex14-GFP puncta that colocalize with the ER (a), do not colocalize with the ER (b), or associate with the ER (c). Stacks of five images with a step of size of 0.25 µm were taken and deconvolved; images from a single plane are shown. Bar, 3 µm. (B) Quantification of the experiment in A; colocalize = (a) + (c) and do not colocalize = (b). At least 50 Pex14-GFP punctae were counted for each strain (mean ± SE; n = 3; **, P < 0.05). (C) Immunogold EM images (I–III) of pex3atg1Δ cells expressing endogenously tagged Pex14-GFP using anti-GFP antibodies. Ia and IIa are cartoon depictions of I and II, respectively. Red arrowheads indicate the gold particle; green arrows, cell wall (CW); blue arrows, plasma membrane (PM); yellow arrows, ER. (D) Time-lapse FM images of pex3atg1Δ cells expressing ss-RFP-HDEL and GFP-Pex14 under the GAL1 promoter. Cells were precultured in a medium with 2% raffinose, induced with 2% galactose for 15 min, shifted back to a medium with only 2% raffinose, and cells were visualized after 15 min (time = 0). Stacks of 10 images with a step of size of 0.25 µm were taken and deconvolved. Images from the indicated plane (z) are shown. Bar, 1 µm. (E) Immuno-EM of cells from D. Cells were grown as in D for 60 min after shifting back to medium with only 2% raffinose. Red arrowheads indicate gold particle.
Figure 7.
Figure 7.
Pex30-2xmCherry is enriched at sites of PPV production. (A) Velocity of Pex14-GFP punctae; mean ± SE of 70 cells. *, P < 0.05. (B) Fluorescence microscopy images of pex3atg1Δ cells expressing endogenous Pex30-2xmCherry and either Pex14-YFP or Sec13-GFP. Stacks of three images with a step of size of 0.3 µm were taken and deconvolved; images from the indicated plane (z) are shown. (C) Quantification of experiment in B. n, number of Pex14-GFP or Sec13-GFP punctae. (D) Cells were grown as in Fig. 6 D, and stacks of three images with a step of size of 0.25 µm were taken at the indicated times and deconvolved. Images were used to quantify percent colocalization of GFP-Pex14 with Pex30-mCherry. n, number of GFP-Pex14 punctae counted. (E) Endogenous Pex14-GFP protein level of pex3atg1Δ cells grown in raffinose and protein level of GFP-Pex14 induced by galactose for 15 min and grown in raffinose for indicated time points.
Figure 8.
Figure 8.
Pex30p and Pex31p regulate the morphology of PPVs. (A) Immunogold EM images of pex3atg1Δ cells expressing endogenous Pex14-GFP using anti-GFP antibodies. (B) As in A, except with pex30pex31pex3atg1Δ cells. Examples of two types of PPV morphology shown: large vesicles (I) and clustered vesicles (II). (C) Quantification of diameter of nonclustered PPVs identified by anti-GFP immuno-EM on cells expressing Pex14-GFP using anti-GFP antibody (mean ± SE in 20 cells; ***, P < 0.005). (D) Immunogold EM images of pex3atg1Δ cells expressing endogenous Pex14-GFP and overexpressing Pex30p using anti-GFP and anti-Pex30p antibodies (red arrowheads, Pex14-GFP; yellow arrowheads, Pex30p). CW, cell wall; N, nucleus; P, preperoxisomal vesicle; V, vacuole.
Figure 9.
Figure 9.
Proposed model of PPV biogenesis from ER subdomains generated by Pex30p and Pex31p.

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References

    1. Agrawal G., and Subramani S. 2016. De novo peroxisome biogenesis: Evolving concepts and conundrums. Biochim. Biophys. Acta. 1863:892–901. 10.1016/j.bbamcr.2015.09.014 - DOI - PMC - PubMed
    1. Agrawal G., Joshi S., and Subramani S. 2011. Cell-free sorting of peroxisomal membrane proteins from the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA. 108:9113–9118. 10.1073/pnas.1018749108 - DOI - PMC - PubMed
    1. Agrawal G., Fassas S.N., Xia Z.J., and Subramani S. 2016. Distinct requirements for intra-ER sorting and budding of peroxisomal membrane proteins from the ER. J. Cell Biol. 212:335–348. 10.1083/jcb.201506141 - DOI - PMC - PubMed
    1. Anwar K., Klemm R.W., Condon A., Severin K.N., Zhang M., Ghirlando R., Hu J., Rapoport T.A., and Prinz W.A. 2012. The dynamin-like GTPase Sey1p mediates homotypic ER fusion in S. cerevisiae. J. Cell Biol. 197:209–217. 10.1083/jcb.201111115 - DOI - PMC - PubMed
    1. Bartlett G.R. 1959. Phosphorus assay in column chromatography. J. Biol. Chem. 234:466–468. - PubMed

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