The Membrane Remodeling Protein Pex11p Activates the Gtpase Dnm1p During Peroxisomal Fission

The membrane remodeling protein Pex11p activates the GTPase Dnm1p during peroxisomal fission

Chris Williamsa, Lukasz Opalinskia,b,1, Christiane Landgrafc, Joseph Costellod, Michael Schraderd, Arjen M. Krikkena,b, Kèvin Knoopsa, Anita M. Krama,b, Rudolf Volkmerc,e, and Ida J. van der Kleia,b,2

aMolecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute, and bKluyver Centre for Genomics of Industrial Fermentation, University of Groningen, 9747 AG Groningen, The Netherlands; cInstitut für Medizinische Immunologie, Charité-Universitätsmedizin Berlin, 10115 Berlin, Germany; dCollege of Life and Environmental Sciences, Biosciences, University of Exeter, Exeter EX4 4QD, United Kingdom; and eLeibniz-Institut für Molekulare Pharmakologie, 13125 Berlin, Germany

Edited by Jennifer Lippincott-Schwartz, National Institutes of Health, Bethesda, MD, and approved April 14, 2015 (received for review October 9, 2014)

The initial phase of peroxisomal fission requires the peroxisomal Fis1p and (in S. cerevisiae) the accessory proteins Mdv1p and membrane protein Peroxin 11 (Pex11p), which remodels the mem- Caf4p (12). Interestingly these proteins are also responsible for brane, resulting in organelle elongation. Here, we identify an ad- mitochondrial fission in yeast (13). ditional function for Pex11p, demonstrating that Pex11p also plays Dnm1p (Drp1 in mammals) (11, 14) is a large GTPase that a crucial role in the final step of peroxisomal fission: dynamin-like achieves membrane fission by forming oligomeric, ring-like struc- protein (DLP)-mediated membrane scission. First, we demonstrate tures around constricted sites on organelle membranes (15). Powered that yeast Pex11p is necessary for the function of the GTPase by GTP hydrolysis, these ring-like structures then tighten further Dynamin-related 1 (Dnm1p) in vivo. In addition, our data indicate until the membrane severs. Interestingly, Dnm1p is recruited to that Pex11p physically interacts with Dnm1p and that inhibiting Pex11p-enriched elongated peroxisomal membranes, suggesting this interaction compromises peroxisomal fission. Finally, we dem- that Pex11p and Dnm1p are functionally linked (16, 17). onstrate that Pex11p functions as a GTPase activating protein Here, we identify a previously unknown role for Pex11p in per- (GAP) for Dnm1p in vitro. Similar observations were made for oxisomal fission. We show that Pex11p directly interacts with Dnm1p mammalian Pex11β and the corresponding DLP Drp1, indicating and that this interaction stimulates the GTPase activity of Dnm1p, establishing Pex11p as a GTPase activating protein (GAP) that plays that DLP activation by Pex11p is conserved. Our work identifies CELL BIOLOGY a crucial role in the last step of the peroxisome fission process. a previously unknown requirement for a GAP in DLP function. Results dynamin-like protein | organelle fission | GTPase activating protein | DLP | Pex11p Is Required for Dnm1p-Mediated Peroxisomal Fission. De- GAP letion of PEX11 or DNM1 in the yeast Hansenula polymorpha affects peroxisome fission (18), resulting in strongly reduced eroxisomes are ubiquitous, single-membrane–bounded cell peroxisome numbers relative to wild-type (WT) controls (17, 19). Porganelles that harbor enzymes involved in a large number Furthermore, in budding H. polymorpha dnm1Δ cells, the single of metabolic processes. Common functions are the β-oxidation enlarged peroxisome in the mother cell forms a long tubular of fatty acids and hydrogen peroxide metabolism. Specialized extension that protrudes into the bud (17), supporting the con- functions include the metabolism of various carbon and organic clusion that fission is blocked at the final step. These extensions nitrogen sources in fungi and the production of plasmalogens are not observed in H. polymorpha pex11Δ cells, in line with the and bile acids in mammals, to name but a few (1). Their impor- model that Pex11p catalyzes the elongation step. To assess tance is underlined by the severe, often lethal human disorders whether Pex11p is essential for Dnm1p-mediated peroxisome caused by defects in peroxisome biogenesis or metabolism (2). Importantly, defects in peroxisome multiplication, caused by mu- Significance tations in genes that control peroxisome fission, also result in severe human disorders (3, 4). Peroxisomal fission is crucial for cell viability because peroxi- Based on data from yeast and mammals, the current model some fission defects cause severe disease. The initial step in for peroxisomal fission describes a three-step process, consisting peroxisomal fission, membrane elongation, requires the mem- of (i) organelle elongation, (ii) constriction, and (iii) the actual brane remodeling protein Peroxin 11 (Pex11p). Here, we iden- scission step (5–7). So far, Peroxin 11 (Pex11p), a highly conserved and abundant peroxisomal membrane protein, is the only tify an additional function for Pex11p, demonstrating that protein known to play a crucial role in the first step (8). Its vital Pex11p also plays a crucial role in the final step of peroxisomal role in peroxisome multiplication is illustrated by the observation fission: membrane separation. We show that Pex11p functions that in all organisms studied so far, Pex11p overproduction re- as a GTPase activating protein (GAP) for Dynamin-related 1 sults in enhanced peroxisome proliferation, whereas PEX11 de- (Dnm1p) and that this GAP activity is conserved from yeast to letion causes a decrease in number, together with an increase in mammalians. This work identifies a previously unknown re- peroxisome size (8). The function of Pex11p in organelle elon- quirement for a GAP in dynamin-like protein function. gation is mediated by the extreme N-terminal region of Pex11p, which can adopt the structure of an amphipathic helix, which Author contributions: C.W., L.O., R.V., and I.J.v.d.K. designed research; C.W., L.O., C.L., upon insertion into membranes induces their curvature, resulting J.C.,M.S.,A.M.Krikken,K.K.,andA.M.Kramperformedresearch;C.W.,L.O.,M.S., A. M. Krikken, K.K., A. M. Kram, R.V., and I.J.v.d.K. analyzed data; and C.W. and I.J.v.d.K. in organelle tubulation (9). wrote the paper. The molecular mechanisms of peroxisome constriction are poorly understood. In contrast, several proteins required for the The authors declare no conflict of interest. final stage of the fission process are known. The first protein This article is a PNAS Direct Submission. shown to be involved in this process was Saccharomyces cer- Freely available online through the PNAS open access option. evisiae Vps1p, a dynamin-like protein (DLP) (10). Later studies 1Present address: Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie revealed that in this organism the DLP Dynamin-related 1 und Molekulare Zellforschung Universität Freiburg, 79104 Freiburg, Germany. (Dnm1p) is also involved in peroxisome fission, especially under 2To whom correspondence should be addressed. Email: [email protected]. peroxisome-inducing growth conditions (11). Dnm1p forms a This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. fission machinery together with the tail-anchored fission protein 1073/pnas.1418736112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1418736112 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 fission, we asked if the peroxisome fission defect of pex11Δ cells organelles. To study whether these structures also still adhered to could be restored by overproducing Dnm1p. Electron microscopy the mother organelle, we performed electron tomography. This (EM) analysis of thin sections revealed increased numbers of per- revealed the presence of numerous constricted tubules (average oxisomal profiles upon overproduction of Dnm1p in WT cells (Fig. diameter, 30 ± 4nm;n = 13) connecting individual vesicular 1andSI Appendix,Fig.S1A), confirming our previous observation structures (Fig. 2 and Movie S1), confirming that the peroxisomal that Dnm1p overproduction in WT cells stimulates peroxisome profiles observed in the dnm1Δ strain overproducing Pex11p are proliferation (20). However, no increase in peroxisomal profiles was still interconnected. Hence, the total number of peroxisomes per observed in thin sections of pex11Δ cells overproducing Dnm1p cell does not increase upon overproduction of Pex11p, although the relative to pex11Δ controls (Fig. 1 and SI Appendix,Fig.S1A), in- number of peroxisomal profiles is enhanced in thin sections of these dicating that in these cells peroxisome fission was not enhanced. cells. Taken together, these data establish that Pex11p is essential This conclusion was confirmed by the quantification of average for Dnm1p-mediated peroxisome fission. numbers of peroxisomes per cell using serial sectioning in pex11Δ and pex11Δ cells overproducing Dnm1p (SI Appendix,Fig.S1B). Pex11p Interacts with Dnm1p Both in Vivo and in Vitro. Previous data Previous 3D reconstruction of serial sections of H. polymorpha from mammalian cells suggested that Pex11β binds indirectly to dnm1Δ cells revealed that at the base of the peroxisome extension the Dnm1p homolog Drp1 (14). To gain further insight into the of the single enlarged organelle, small vesicular extensions are relationship between Pex11p and Dnm1p in H. polymorpha,we formed, which still adhere to the mother organelle (21) (Fig. 1). subjected lysates of WT, pex11Δ, and dnm1Δ cells to coimmu- This explains the presence of multiple peroxisomal profiles in thin noprecipitation analysis using anti-Dnm1p antibodies. As shown sections of dnm1Δ cells. EM analysis of thin sections of dnm1Δ cells in Fig. 3A, Pex11p can be coprecipitated with Dnm1p antibodies overproducing Pex11p revealed an increase in peroxisomal profiles from WT cell lysates, but not from lysates from both mutant per cell section (Fig. 1 and SI Appendix,Fig.S1A). These profiles strains, indicating that both proteins interact in vivo. Taking our were smaller than the mother peroxisomes in dnm1Δ cells but much analysis further, we used purified Dnm1p from E. coli, together dnm1Δ larger than the vesicular structures that associate with with Pex11-His6, isolated from yeast (Fig. 3B), to demonstrate that the Dnm1p–Pex11p interaction is direct (Fig. 3C).

Large, Hydrophobic Residues in the N Terminus of Pex11p Control WT WT + DNM1 Dnm1p Binding. To identify regions in Pex11p that are involved in Dnm1p binding, we performed a peptide blot analysis using overlapping 12-mer peptides from the N-terminal 134 amino acids of Pex11p and purified His6GST-Dnm1p (Fig. 4A). This re- M P gion of Pex11p was deemed most likely to bind Dnm1p because it P is exposed to the cytosol (SI Appendix,Fig.S2). Our peptide blot – V M analysis identified four potential binding sites (termed B1 B4) for A N His6GST-Dnm1p in Pex11p (Fig. 4 , boxed). These regions did N not display binding to His6GST alone (Fig. 4A). To determine which residues control the Dnm1p–Pex11p interaction, a peptide array was produced, where each residue of the interacting regions B1–B4 was substituted with every other amino Δpex11 Δpex11 + DNM1 acid. These peptides were spotted onto cellulose membranes, and the ability of these peptides to bind His6GST-Dnm1p was tested M (Fig. 4 B–E). Introduction of alternative amino acids into the M peptides covering regions B2 (Fig. 4C)andB4(Fig.4E) did not N significantly disturb the ability of His6GST-Dnm1p to bind, which P suggests that either these regions bind nonspecifically or no single amino acid substitution can disturb binding. In contrast, the peptides covering regions B1 and B3 were sensitive to single N B D V P amino acid substitutions (Fig. 4 and ). Specifically, changing V Leu12, Leu15, Ile16, Phe18, or Leu19 in B1 or Leu59, Leu62, or Phe63 in B3 into Alanines inhibited the interaction. These residues are largely conserved within the Pex11p family (SI Appen- dix, Fig. S3B). Furthermore, introduction of a Proline into B1 Δdnm1 Δdnm1 + PEX11 also inhibited the interaction, suggesting that Dnm1p binding requires an intact alpha helix. Secondary structure predictions propose that residues 14–19 of Pex11p are indeed alpha helical, M whereas the B3 region is part of a larger amphipathic helix that M extends from residue 56–86 (SI Appendix, Fig. S3A). N P Mutations in the DLP Binding Site of Pex11p Inhibit Peroxisome V Fission in Yeast and Mammals. Disturbances to peroxisomal fission P result in lower numbers of peroxisomes per cell (17, 19). Therefore, we determined the effect of inhibiting the Pex11p–Dnm1p interaction on peroxisome numbers in H. polymorpha cells using PMP47-GFP as a peroxisomal membrane marker and fluorescence microscopy (Fig. 5). The Pex11p–Dnm1p interaction was inhibited Fig. 1. Pex11p is required for Dnm1p-mediated peroxisomal fission. by mutating the Leucine residues at positions 15 and 59 in Pex11p + to Alanines (L15A and L59A), mutations which disturbed the H. polymorpha WT cells (WT), WT cells overproducing Dnm1-GFP (WT DNM1), – pex11Δ cells (Δpex11), pex11Δ cells overproducing Dnm1-GFP (Δpex11 + DNM1), Dnm1p Pex11p interaction (Fig. 4). Expression of the L15A or dnm1Δ cells (Δdnm1), or dnm1Δ cells overproducing Pex11p (Δdnm1 + PEX11) L59A forms of Pex11p in pex11Δ cells did not compromise perox- were grown for 16 h on methanol-containing media and analyzed by EM. isome fission (Fig. 5B). However, the two putative Dnm1p binding (Scale bars, 1 μm.) M, mitochondria; N, nucleus; P, peroxisome; V, vacuole. Arrow sites in Pex11p display similar amino acid sequences, as well as represents the peroxisomal vesicle associated with the mother organelle. similar secondary structural properties (SI Appendix,Fig.S3), which

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1418736112 Williams et al. Downloaded by guest on October 2, 2021 A The Amphipathic Helix of Pex11p Functions as a GAP for Dnm1p in 180° Vitro. GTP hydrolysis by Dnm1p is needed to achieve organelle fission (11, 16, 24). Because Pex11p is required for Dnm1p P1 P1 P1 function, we evaluated whether Pex11p binding alters the kinetic P4 properties of Dnm1p. Purified Dnm1p (Fig. 7A) was able to P2 P3 hydrolyze GTP in a time-dependent manner (Fig. 7B) and dis- − played a catalytic activity (Kcat) of 2.16 ± 0.42 min 1 and a K ± μ D B substrate affinity ( m) of 381 6.5 M (Fig. 7 ). These values P1 are similar to those of mammalian Drp1, isolated from S. cer- P1 P1 evisiae (25). The addition of purified Pex11-His6 resulted in a small increase in the GTPase activity of Dnm1p (SI Appendix, Fig. S5). However, addition of detergents to Dnm1p, which were P2 P4 essential to solubilize full-length Pex11-His6, strongly reduced its P3 activity (SI Appendix, Fig. S5), which hampered the analysis of the effect of full-length Pex11-His6 on Dnm1p. Therefore, we C continued our analysis using peptides of Pex11p. Peptides cor- P1 responding to the binding regions B1 or B3 in Pex11p did not P1 P1 alter the activity of Dnm1p (SI Appendix, Fig. S6A). However, the B3 binding site resides in a larger amphipathic helix, ex- tending from residues 56 to 86 (SI Appendix, Fig. S2). Addition of the complete amphipathic helix to Dnm1p significantly en- P2 P4 − P3 hanced the catalytic activity (Kcat = 4.14 ± 0.32 min 1) as well as substrate affinity (Km = 237 ± 13 μM). This stimulation was also observed when reactions were performed in the presence of high Fig. 2. Pex11p overproduction in dnm1Δ cells remodels peroxisomes into SI Appendix B interconnected compartments. H. polymorpha dnm1Δ cells overproducing salt ( , Fig. S6 ), conditions known to block Dnm1p Pex11p were grown for 16 h on methanol-containing media and analyzed by oligomerization (25). No increase in activity was observed when electron tomography. (A) Surface rendering of four adjacent peroxisomes a similar peptide was added where Leu59, Leu62, and Phe63 (P1–P4). (B) The 5.5-nm-thick digital slices illustrating the neck-like tubules were altered to Alanine residues (SI Appendix, Fig. S6C). This (arrows) that connect the indicated peroxisomes. Slices have been slightly CELL BIOLOGY tilted through the tomographic volume at the indicated position in A to maximize clarity. (C) Schematic drawing of the peroxisomal membranes shown in B. [Scale bars, (A) 100 nm and (B and C) 25 nm.] A Δ B WT dnm1 pex11Δ 12

could suggest a level of redundancy. To investigate this, we pro- 130 - - Dnm1p 100 - duced a version of Pex11p where both Leu15 and Leu59 were 100 - 70 - mutated to Alanines (L15A/L59A) (Fig. 5). pex11Δ cells producing Co-IP 55 - this mutant contained significantly reduced average numbers of - Pex11p 40 - peroxisomes per cell (Fig. 5A and SI Appendix,TableS1), as well as 25 - 35 - an increased percentage of cells with no peroxisomes or one per- 130 - - Pyc oxisome, typical for pex11Δ cells (SI Appendix,Fig.S4A). As 25 - expected, Pex11 L15A/L59A displayed a reduced ability to bind Input - Dnm1p Dnm1pinvitro(SI Appendix,Fig.S4B). Introduction of the L15A/ 100 - L59A mutations into Pex11p did not alter the levels of Pex11p (Fig. 5C) nor compromise the ability of the strain expressing this mutant - Pex11p to grow on methanol (SI Appendix,Fig.S4C), indicating that the 25 - observed fission defect results from disturbing the Pex11p–Dnm1p C interaction. As in H. polymorpha, peroxisome fission in mammals requires the action of a DLP, the Dnm1p homolog Drp1 (22). Sequence Input Dnm1pInput ColumnFlow throughElution

alignment analysis indicates that many of the residues that reg- + His6GST-Pex2p ulate the Dnm1p–Pex11p interaction in yeast are conserved in αDnm1p mammalian Pex11β, whereas the domain structure of Pex11β is + Pex11-His6 also highly similar (SI Appendix, Figs. S3 and S7). To investigate the link between Drp1 and Pex11β further, we mutated the resi- αGST + His GST-Pex2p dues corresponding to Leu15 and Leu59 in mammalian Pex11β 6 (Trp4 and Leu48) and followed the effect on peroxisome fission in α + Pex11-His COS-7 cells (Fig. 6). Expression of Pex11β L48A induced peroxi- Pex11p 6 some proliferation in a similar manner to WT Pex11β (23). Con- versely, altering Trp4 of Pex11β to an Alanine (W4A) inhibited Fig. 3. Pex11p interacts with Dnm1p both in vivo and in vitro. (A) Coimmuno- peroxisome fission and resulted in the formation of hypertubular precipitation analysis (Upper panels) of cell lysates (Lower panels) derived from peroxisomes (Fig. 6), a sign that Drp1 activity is inhibited (22). WT, pex11Δ,anddnm1Δ cells, using antibodies raised against H. polymorpha Immunofluorescence studies demonstrated that Drp1 localization is Dnm1p. Samples were analyzed by SDS/PAGE and Western blotting using anti- not altered by these mutations (SI Appendix,Fig.S8A). Signifi- bodies raised against Pex11p, Dnm1p, and Pyruvate carboxylase (Pyc, loading cantly, introduction of the W4A mutation into a peptide derived control). (B) SDS/PAGE and coomassie staining (1) and Western blot probed with Pex11p antibodies (2) of Pex11-His purified from yeast. (C) Pull-down assays using from Pex11β disturbedtheabilityofDrp1tobindtothispeptidein 6 SI Appendix B Dnm1p purified from E. coli with purified Pex11-His6 or His6GST-Pex2 really in- vitro ( ,Fig.S8 ). These observations support the teresting new gene (RING) domain bound to Ni-NTA resin. Samples were analyzed conclusion that, similar to yeast, Drp1-mediated peroxisomal fission by SDS/PAGE and Western blotting using antibodies raised against S. cerevisiae can be compromised by disturbing the Drp1–Pex11β interaction. Dnm1p, GST, and Pex11p. Equal portions were loaded per lane.

Williams et al. PNAS Early Edition | 3of6 Downloaded by guest on October 2, 2021 directly with Dnm1p, plus that this interaction is crucial for per- A + His p1mnD-TSG- siH+ -GST 6 6 oxisomal fission, establishes that Pex11p performs an additional 1 B1 20 1 B1 B2 20 21 40 21 B2 40 yet vital role in the last stage of peroxisomal fission. How can these B3 41 60 41 B3 60 61 80 61 80 data be integrated into the prevailing model describing peroxi- 81 100 81 100 101 B4 120 101 B4 120 somal fission? The binding sites for Dnm1p reside in the N-ter- 121 121 α-His minal region of Pex11p, as does the membrane remodeling 6 α-His 6 amphipathic helix of Pex11p (9). Furthermore, one Dnm1p binding B wTAt C DYEG I QPNMLKHF RS V W C wTAt C DYEG I QPNMLKHF RS V W site (B3, residues 55–70) is present in this amphipathic helix, 10 -P 34 - Y T V suggesting the existence of a synergistic relationship between the L T T K membrane remodeling and GTPase activating roles of Pex11p. K L L L Our previous data establish that this amphipathic helix adopts an I A N Y unstructured conformation in aqueous solution (9), suggesting F Y L L that this region of Pex11p can undergo transition from an un- E L T R folded to helical conformation. This transition may facilitate N T N G insertion of the amphipathic helix into the peroxisomal mem- G S R brane, to initiate fission. As the introduction of helix-breaking D S K V 51 - N mutations can disturb the Dnm1p interaction with Pex11p peptides 28 -L (Fig. 4), we conclude that helix formation is required for Pex11p to D E bind Dnm1p. Hence, Dnm1p binding, and consequently stimula- wTAt C DYEG I QPNMLKHF RS V W wTAt C DYEG I QPNMLKHF RS V W tion of Dnm1p GTPase activity, is likely to occur after Pex11p has 55 -L 114 -T V V remodeled the peroxisomal membrane, suggesting a spatiotempo- R H R W ral relationship. On the other hand, our data on mammalian L L Q K β β D L Pex11 ,whichsuggestthatadifferentregionofPex11 binds to L L SI Appendix F G and stimulates the activity of Drp1 (Fig. 6 and ,Figs.S7 T L L L and S8), indicate that although the GAP activity of Pex11p is con- S R R N served in several species, the underlying mechanisms may differ. K R S. cerevisiae P N In several Dnm1p binding partners are known 70 -L G 130 -K to regulate peroxisomal fission, including the membrane protein Fis1p and the cytosolic proteins Mdv1p and Caf4p (12, 27). Fig. 4. Detailed analysis of the Pex11p–Dnm1p interaction. (A)Peptideblots Originally these proteins were proposed to facilitate Dnm1p re- of overlapping 12-mer peptides covering the N-terminal 134 amino acids of cruitment to membranes, yet recent data on mitochondrial fission Pex11p were incubated with His6GST-Dnm1p (Left panel) or His6GST (Right identified additional, postrecruitment roles. Importantly, these panel). Bound protein was detected using antibodies raised against the His6 binding partners do not stimulate Dnm1p GTPase activity, instead tag. The four potential interacting regions, corresponding to amino acids 10– appearing to modulate Dnm1p oligomer assembly (25). In mam- 29 (B1), 35–51 (B2), 55–70 (B3), and 113–130 (B4), are boxed. (B–E) Substitution – mals Drp1 binds not only to Fis1p (28) but additionally to the analysis with an array of Pex11p peptide variants derived from residues 10 28 membrane protein Mff (29), and although the role of the Drp1– (B), 34–51 (C), 55–70 (D), and 114–130 (E). Each spot corresponds to a version of Fis1p interaction in organelle fission remains unclear, binding to the WT peptide (sequence shown in the left margin; the first and last amino acid are numbered) where one amino acid was changed to one of the 20 Mff allows Drp1 to associate with organellar membranes (30). amino acids (shown across the top of the blot). Spots in the first column rep- Significantly, Mff binding does not enhance the GTPase activity resent replicas of the WT sequence. Peptides were spot-synthesized on cellu- of Drp1 (25). Consequently, the available data on DLP interacting

lose membranes, incubated with His6GST-Dnm1p, and probed as above. proteins suggest that DLP-mediated organelle fission is coordinated Substitutions that disturb binding (B and D)areboxed.

peptide is unable to bind to Dnm1p in vitro (SI Appendix, Fig. A WT pex11Δ L15A/L59A S6D), indicating that the interaction between the amphipathic helix and Dnm1p is crucial for the observed stimulation. Finally, we established that the peptide from Pex11β that is able to bind to Drp1 (SI Appendix, Fig. S8) can also stimulate its GTPase activity in vitro (SI Appendix, Fig. S7), clearly demonstrating that Pex11p, in addition to its role in membrane remodeling (9), can function as a GAP for DLPs during peroxisome fission. pex11Δ B 5 C + Discussion 4 Pex11p initiates peroxisomal fission by catalyzing organelle elon- * - WT L15A L59A L15A/L59A gation (9, 26). In the present study, we identify a new role for yeast 3 70 - - PMP47-GFP Pex11pinthefinalstageofperoxisomal fission, namely as a GAP 2

Average No. No. Average 55 - * - EF 1α for Dnm1p. Similar observations were made for the mammalian Peroxisomes/cell 1 β homologs Pex11 and Drp1. 0 35 - - Pex11p-His Our data clearly demonstrate that Pex11p and Dnm1p are re- pex11Δ WT L15A L59A L15A/L59A 25 - 6 quired for different aspects of peroxisomal fission, as the lack of one protein cannot be suppressed by overproduction of the other. Fig. 5. Mutations in the Dnm1p binding sites of Pex11p disturb peroxisome H. polymorpha dnm1Δ fission. (A) Fluorescence microscopy analysis of methanol-grown pex11Δ cells Pex11p overproduction in cells results in Δ Δ tubulation, but not scission, of the peroxisomal membrane, (pex11 )andpex11 cells producing WT Pex11p (WT) or Pex11p L15A/L59A pex11Δ (L15A/L59A). Cells coproduced the peroxisomal membrane marker PMP47-GFP. whereas cells contain a single peroxisome irrespective of (Scale bar, 5 μm.) (B) Quantification of peroxisome numbers in pex11Δ cells whether Dnm1p is overproduced or not. These data fit a model producing WT or mutant forms of Pex11-His6. All strains coexpressed PMP47- where Pex11p and Dnm1p act together in the final step of per- GFP, the readout of which was used to quantify peroxisome numbers. Values oxisomal fission. Pex11p also functions as an initiator of the fission represent the mean ± SD of two independent experiments (*P < 0.05). process through insertion of an amphipathic helix into the per- (C) Western blots of cell lysates from pex11Δ cells producing WT or mutant oxisomal membrane, with Dnm1p-mediated scission of the or- forms of Pex11p, coproducing PMP47-GFP. Blots were probed with antibodies

ganelle following. However, the observation that Pex11p interacts raised against elongation factor (EF) 1α (loading control), GFP, and the His6 tag.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1418736112 Williams et al. Downloaded by guest on October 2, 2021 oligomerization, and based on our data, modulators that stimulate activity during membrane separation. The recent observation that a 30% increase in the activity of a DLP, as measured with in vitro approaches, can cause a signif- icant increase in membrane remodeling capabilities of the protein in vivo (31) supports our observation that a twofold increase in the activity of Dnm1p in vitro translates into efficient peroxisome fission in vivo. Binding of Pex11p to a DLP occurs at its site of action, the peroxisomal membrane, guaranteeing that the resulting increase in GTPase activity occurs at the right time and right place for efficient membrane fission. Leading on from this, recent data indicate that the activity of Drp1 is enhanced by cardiolipin (32). This lipid, which plays a role in mitochondrial fission (33), is also a potent stimulator of membrane tubulation (32). Cardiolipin is present in the peroxisomal membrane (34), whereas the amphipathic helix of Pex11p can only tubulate lipo- somes that contain cardiolipin (9). Taken together, these observations support the suggestion that DLP activity and, consequently, the final act of organelle fission can be fine-tuned by factors on the organelle membrane. Identifying other factors that could control DLP activity in similar ways will provide invaluable insight into how membrane scission is achieved. Our understanding of peroxisomal fission was advanced con- siderably by the observation that Pex11p directly controls membrane elongation (9, 26). In addition, previous work from our group (21) suggests that Pex11p also directs the redistribution of peroxisomal membrane proteins during fission, supporting the idea that the function of Pex11p goes much further than membrane

remodeling. We propose that Pex11p is in fact a master regulator CELL BIOLOGY of peroxisomal fission, providing vital contributions to a range of events that occur during fission. Materials and Methods For details of plasmids, oligonucleotides, and H. polymorpha strains used in this study, see SI Appendix.

Fig. 6. Pex11β W4A results in the accumulation of tubular peroxisomes. Culture Conditions. H. polymorpha cells were grown in batch cultures at 37 °C (A) COS-7 cells were transfected with Pex11β-Myc, Pex11β-L48A-Myc, Pex11β- on mineral media supplemented with 0.25% glucose or 0.5% methanol, W4A-Myc, or Pex11β-W4A/L48A-Myc. Fixed cells were labeled with anti- with or without 0.05% glycerol as the carbon source and 0.25% ammonium Pex14p and anti-Myc antibodies. Higher magnification view of boxed re- sulfate or methylamine as the nitrogen source. Leucine, when required, was gions is shown in the Right panel. (B) Immunoblotting of cell lysates with added to a final concentration of 30 μg/mL COS-7 cells (ECACC 87021302) anti-Myc and anti-Tubulin (loading control) antibodies. (C) Quantitative and was maintained in DMEM supplemented with 10% (wt/vol) FCS (Life β analysis of peroxisome morphology in cells expressing Pex11 mutants at Technologies) at 37 °C in a 5% CO2-humidified incubator. Cells were trans- several time points after transfection. (D) The percentage of cells with fected by electroporation (BTX ECM 630; BTX Instrument Division, Harvard hypertubular peroxisomes (48 h). For quantitative evaluation of peroxisome Apparatus; settings, 230 V, 1500 μF, 125 Ω) as described (22). morphology, peroxisomes were categorized as tubular (<5 μm) or unusually long, hypertubular (>5 μm). Values represent the mean ± SD of at least three Biochemical Techniques. Details on cell extracts, protein expression/purifica- independent experiments (**P < 0.01, ***P < 0.001). (Scale bar, 10 μm.) tion, and binding assays can be found in SI Appendix.

Peptide Blot Assays. The peptide arrays corresponding to the first 134 amino by a range of binding partners that provide different functions at acids of Pex11p were synthesized on amino-modified cellulose membranes different stages of the fission process, including recruitment (β-alanine membrane) according to SPOT synthesis protocols (35). The design factors that govern localization, adaptor proteins that control and the peptide spot arrangement for the substitutional analyses of variant

ADB C

20 ) 3 130 - -1 Dnm1p + Pex11p Amph 100 - Dnm1p + Pex11p Amph 2,5 70 - 15 55 - 2 Dnm1p + Pex11p Amph Dnm1p 40 - -1 10 1,5 Vmax (μM Pi released min ) 1.73 ± 0.34 3.33 ± 0.26 35 - Dnm1p M Pi released min M Pi released 1 Kcat (min-1) 2.16 ± 0.42 4.14 ± 0.32 25 - 5 0,5 Pex11p Amph Km (μM) 381 ± 6.5 237 ± 13

0 Velocity ( 0 15 - 0102030 0 200 400 600 800 1000 1200 Time (min) [GTP] (μM)

Fig. 7. The amphipathic helix of Pex11p functions as a GAP for Dnm1p in vitro. (A) SDS/PAGE and coomassie staining analysis of Dnm1p purified from E. coli. (B) Time course of GTP hydrolysis by Dnm1p (open circles), Dnm1p together with a peptide of the amphipathic helix of Pex11p (closed squares), or the Pex11p peptide alone (open diamonds). (C) Steady-state kinetics of GTP hydrolysis by Dnm1p in the presence (closed squares) and absence (open circles) of the Pex11p amphipathic helix peptide. (D) Kinetic parameters of Dnm1p in the presence and absence of the Pex11p amphipathic helix peptide. Values represent the mean ± SD of three separate measurements.

Williams et al. PNAS Early Edition | 5of6 Downloaded by guest on October 2, 2021 peptides derived from the sequence were carried out using the in-house dnm1Δ overproducing Pex11p were cryo-fixed in liquid ethane using the sand- software LISA. For further details, see SI Appendix. wich plunge freezing method (38). Cells were freeze-substituted in 1% osmium tetroxide, 0.5% uranyl acetate, and 5% (vol/vol) distilled water in acetone GTPase Activity Measurements. The GTP hydrolyzing activity of purified Dnm1p using the fast low-temperature dehydration and fixation method (39). Cells (0.8 μM), in the absence or presence of Pex11p peptides (8 μM), was followed by were infiltrated overnight with Epon and polymerized for 48 h at 60 °C. We measuring the release of inorganic phosphate using the Pi ColorLock Gold kit cut 200-nm-thick sections and overlayed them with 10 nm of fiducial gold (Innova Biosciences) according to the manufacturer’s instructions. For further particles. Two single-axis tilt series, each containing 131 images with 1° tilt details, see SI Appendix.TheVmax,Kcat,andKm were calculated in Microsoft Excel increments, were acquired on a FEI Tecnai20 running at 200 kV using the FEI using nonlinear regression curve fitting. The data presented represent the aver- automated tomography acquisition software and a cooled slow-scan charge- ± age SD of three separate measurements. coupled device camera (Ultrascan 4000; Gatan) in 2 × 2 binned mode with a final pixel size of 1.1 nm. The tilt series were aligned and reconstructed by Fluorescence Microscopy. All yeast fluorescence microscopy images were acquired the simultaneous iterative reconstruction technique (SIRT) algorithm using × using a Zeiss Plan-Neofluar 100 /1.3oilobjective.Confocalimagesofyeastcells the IMOD software package (40) and analyzed using the AMIRA visualiza- were captured using a Zeiss LSM510, using photomultiplier tubes (Hamamatsu tion package (TGS Europe). To generate three-dimensional surface-rendered Photonics); images were acquired using Zen 2009 software. GFP fluorescence models in AMIRA, masks of peroxisomes were drawn manually and after- was analyzed by excitation of the cells with a 488-nm argon ion laser (Lasos), and ward improved by thresh-holding. The movie was shot in AMIRA as se- emission was detected using either a 500–530-nm or a 500–550-nm bandpass quential TIF images, which subsequently were merged into a movie using emission filter. For quantification of peroxisomes, Z stack images were made FFmpeg (www.ffmpeg.org). using an interval of 0.6 μm. Wide-field fluorescence microscopy was performed using a Zeiss Axioscope50 fluorescence microscope. Images were acquired ACKNOWLEDGMENTS. The authors thank E. Boekema for access to advanced with a Princeton Instruments 1300Y CCD camera (Roper Scientific). GFP fluo- electron microscope facilities; R. de Boer, M. Mulder, and T. Schrader for rescence was visualized with a 470/40-nm bandpass excitation filter, a 495-nm technical assistance; L. Bosgraaf for help with the ImageJ plugin; D. Crane dichromatic mirror, and a 525/50-nm bandpass emission filter. Image analysis was for Pex14p antibodies; J. Nunnari for S. cerevisiae Dnm1p antibodies; carried out using ImageJ (rsb.info.nih.gov/nih-image/) and Adobe Photoshop O. Daumke for the Drp1 plasmid and advice; and M. Veenhuis for discussions. (Adobe Systems). For immunofluorescence microscopy on COS-7 cells, cells grown C.W. is supported by Open Programme Grant 821.02.022 and Vidi Grant on glass coverslips were fixed with 4% (vol/vol) paraformaldehyde in PBS pH 723.013.004 from the Netherlands Organisation for Scientific Research (NWO). 7.4, permeabilized with 2.5 μg/mL digitonin, blocked with 1% BSA, and incu- K.K. is supported by European Union Marie Curie Intra-European Fellowship bated with primary and secondary antibodies as described (36). Information on FP7-330150. A. M. Kram is supported by the research program of the Kluyver Centre for Genomics of Industrial Fermentation, as part of the Netherlands quantification and statistical analysis can be found in SI Appendix. Genomics Initiative/NWO. M.S. is supported by Biotechnology and Biological Sciences Research Council Grant BB/K006231/1 and Wellcome Trust Institutional EM and Electron Tomography. For EM, cells were fixed in 1.5% (wt/vol) KMnO4 Strategic Support Award WT097835MF. I.J.v.d.K. and M.S. are supported by and prepared for EM as described (37). For electron tomography, H. polymorpha Marie Curie Initial Training Networks Action PerFuMe Grant 316723.

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