Seipin and the membrane-shaping protein Pex30 cooperate in organelle budding from the endoplasmic reticulum

Wang, Sihui; Idrissi, Fatima-Zahra; Hermansson, Martin; Grippa, Alexandra; Ejsing, Christer S.; Carvalho, Pedro

doi:10.1038/s41467-018-05278-2

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Published: 27 July 2018

Seipin and the membrane-shaping protein Pex30 cooperate in organelle budding from the endoplasmic reticulum

Sihui Wang¹^na1,
Fatima-Zahra Idrissi^2,3^na1,
Martin Hermansson⁴,
Alexandra Grippa^2,3,
Christer S. Ejsing ORCID: orcid.org/0000-0003-4963-0276^4,5 &
…
Pedro Carvalho ORCID: orcid.org/0000-0002-9691-5277^1,2,3

Nature Communications volume 9, Article number: 2939 (2018) Cite this article

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Abstract

Lipid droplets (LDs) and peroxisomes are ubiquitous organelles with central roles in eukaryotic cells. Although the mechanisms involved in biogenesis of these organelles remain elusive, both seem to require the endoplasmic reticulum (ER). Here we show that in yeast the ER budding of these structurally unrelated organelles has remarkably similar requirements and involves cooperation between Pex30 and the seipin complex. In the absence of these components, budding of both LDs and peroxisomes is inhibited, leading to the ER accumulation of their respective constituent molecules, such as triacylglycerols and peroxisomal membrane proteins, whereas COPII vesicle formation remains unaffected. This phenotype can be reversed by remodeling ER phospholipid composition highlighting a key function of these lipids in organelle biogenesis. We propose that seipin and Pex30 act in concert to organize membrane domains permissive for organelle budding, and that may have a lipid composition distinct from the bulk ER.

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Introduction

The endoplasmic reticulum (ER) produces large amounts of phospholipids and membrane proteins to supply most cellular organelles. Upon their synthesis, many of these molecules are packaged into COPII-coated vesicles to traffic to their final destination¹. Although the mechanisms of COPII vesicle formation are well characterized, how specific proteins and lipids in the ER give rise to other organelles, such as lipid droplets (LDs) and peroxisomes, remains largely unknown². These are ubiquitous organelles and mutations impairing their functions result in devastating diseases^3,4. Despite their importance in cell physiology, LD and peroxisome biogenesis at the surface of the ER remain mysterious².

In yeast, de novo peroxisome biogenesis involves the budding of membrane-bound pre-peroxisomal vesicles (PPVs) from specific ER domains distinct from COPII assembly sites^5,6. As cargo, these vesicles were shown to contain certain peroxisomal membrane proteins^5,7,8,9, such as Pex3, Pex15, and the importomer complex component Pex14, required for the translocation of soluble proteins from the cytoplasm into the peroxisomal matrix¹⁰. PPV-budding sites are enriched in the membrane-shaping protein Pex30 but their exact composition and mechanism of formation are unknown⁵. In fact, it has been suggested that formation of functional peroxisomes results from the fusion of two distinct types of PPVs formed at the ER^11,12. However, this latest model is controversial and how PPVs mature to become functional peroxisomes remains ill-defined^2,3,13.

LDs are structurally distinct from peroxisomes consisting of a neutral lipid core, mainly of triacylglycerols (TAGs) and steryl esters, encased in a unique phospholipid monolayer instead of the typical bilayer present in most organelles¹⁴. The biogenesis of LDs also occurs at specific ER domains but whether and how these relate to PPV-budding sites have not been explored. According to the prevailing model, neutral lipids between bilayer leaflets at specific ER sites coalesce into a lens. The growth of the lens by addition of more neutral lipids results in LD budding into the cytoplasm^14,15,16. The growth of a LD at the ER surface is also essential for targeting hairpin-containing membrane proteins to the LD monolayer¹⁷. Curiously, the LD targeting of at least some hairpin-containing proteins requires factors involved in peroxisomal membrane protein targeting¹⁸, suggesting a link between LD and peroxisome biogenesis.

LD budding sites are marked by seipin, a widely conserved protein frequently mutated in familial forms of lipodystrophy^19,20,21. In agreement with its central role at the ER-LD interface, mutations in seipin lead to aberrant LD biogenesis, morphology, and dynamics in a variety of cell types^{4,20,21,22,23,24,25}. Abnormal ER-LD contacts in seipin mutants also reduce efficient targeting of LD-specific proteins and increase the packing defects in the LD monolayer, as detected by the unspecific recruitment of amphipathic helix-containing proteins^22,23,25. However, factors that cooperate with seipin have not been identified and, as in the case of PPVs, the precise composition of LD budding sites is unclear.

Here we identify Pex30 as a factor cooperating with seipin in the biogenesis of both LDs and PPVs from the ER. We show that Pex30 and seipin are enriched at ER domains where both LDs and PPVs appear to form. Consistent with a functional role at those ER domains, simultaneous deletion of Pex30 and seipin results in strong impairment of LD and peroxisome biogenesis. Strikingly, the organelle budding defect in these mutants can be largely restored by remodeling ER phospholipid composition. These data suggest that seipin and Pex30 organize membrane domains with a lipid composition distinct from the bulk ER, and that is permissive for organelle budding.

Results

Pex30 and seipin cooperate during LD biogenesis

To identify factors that cooperate with seipin, we hypothesized that they would compensate for its loss and be enriched at LD budding sites in seipin mutants. To test this possibility, fractions enriched in LDs containing also remnants of adjacent but not bulk ER membranes, were isolated from wild-type (WT) and seipin mutant cells, and analyzed by quantitive proteomics²². In yeast two polypeptides, Fld1 and Ldb16, form a functional seipin complex²⁵. We found that Pex30 was strongly enriched in fractions isolated from fld1∆ and ldb16∆ cells (Table 1 and Supplementary Tables S1, S2). Other abundant ER proteins were not as enriched in these fractions, suggesting that the effect on Pex30 was specific²². To validate the proteomics result, we investigated the localization of functional, endogenously expressed Pex30 fused to the fluorescent protein monomeric Neon Green (Pex30-mNG). In WT cells, Pex30-mNG localized non-uniformly throughout the ER as detected by Sec63-mCherry (Fig. 1a), as previously reported^5,6. In agreement with the proteomics data, fld1∆ and ldb16∆ cells showed dramatic enrichment of Pex30-mNG in LD proximal regions (Fig. 1a and Supplementary Figure 1). This enrichment was observed during all growth stages and was not a consequence of increased Pex30 protein levels, as these were comparable between seipin mutant and WT cells (Fig. 1b). Thus, in the absence of seipin, Pex30 specifically accumulates at ER-LD contacts.

Table 1 Pex30 is enriched in LDs isolated from fld1∆ and ldb16∆ cells

Full size table

To dissect the links between seipin and Pex30 we combined deletions on both genes. Remarkably, both pex30∆fld1∆ and pex30∆ldb16∆ grew much slower than WT cells and the individual deletion mutants. The growth defect was observed at 30 °C and much exacerbated at lower (25 °C) and higher (37 °C) temperatures, both in rich (Fig. 1c) and defined media (Supplementary Figure 2a). Pex30 is the founding member of a larger family of membrane-shaping proteins also including Pex28, Pex29, Pex31, and Pex32^5,6,26,27. However, mutants with combined deletion of seipin and any other members of the Pex30 family grew mostly at normal rates (Supplementary Figure 2b). Thus, within this protein family, Pex30 appears to have some unique function. Moreover, cells with seipin deletion in conjunction with genes essential for peroxisome biogenesis, such as Pex3 and Pex19, also grew at normal rates, indicating that the defect in pex30∆fld1∆ or pex30∆ldb16∆ was not due to a general loss of peroxisomes (Supplementary Figure 2c). Together, these data indicate that enrichment of Pex30 at ER-LD contacts in seipin mutants is functionally relevant.

Pex30 was recently shown to be involved in the budding of PPVs from the ER^5,6. Given its enrichment at ER-LD contacts in seipin mutants, we wondered whether it could also contribute to LD budding. As previously described^20,21,22,25, fld1∆ and ldb16∆ mutants displayed abnormally supersized or aggregated LDs, as detected by fluorescence microscopy of cells stained with the neutral lipid-specific dye Bodipy (Fig. 2a). In contrast, LDs in pex30∆ mutants appeared largely indistinguishable from WT cells. However, it should be noted that 4% of pex30∆ cells (n = 300) showed additional Bodipy-positive structures (Supplementary Figure 3a). These were never detected in WT, fld1∆, and ldb16∆ cells, and were invariably less bright than LDs and reminiscent of small membranous regions, a hypothesis supported by electron microscopy (EM) (Supplementary Figure 3b). To further explore a potential cooperation between seipin and Pex30, we analyzed the double mutants pex30∆fld1∆ and pex30∆ldb16∆. Both mutants showed a unique dispersed Bodipy pattern of variable size but often occupying a large fraction of cellular area (Fig. 2a, b). Such aberrant staining was detected in the majority of double mutant cells (Fig. 2b). Moreover, the dispersed Bodipy pattern was frequently scattered with brighter spots of variable sizes. Electron microscopy characterization of the double mutants suggested that the dispersed Bodipy-positive structures correspond to highly convoluted ER membranes (Fig. 2c). Consistent with their staining by Bodipy, these membranous structures are likely enriched in neutral lipids, which do not bind the EM staining reagent uranyl acetate and appear electron-translucent. Importantly, classical LD-like structures were virtually absent from the double mutant cells. The ER origin of the convoluted membranes present in pex30∆fld1∆ and pex30∆ldb16∆ cells was confirmed by fluorescence microscopy in cells expressing endogenous levels of the ER marker protein Sec63-mNG (Fig. 2d). Shotgun lipidomics analysis^28,29 revealed that, in relation to WT or any of the single mutants, pex30∆fld1∆ or pex30∆ldb16∆ had a dramatic phospholipid surplus consisting essentially of phosphatidylcholine (PC) and phosphatidylinositol (PI), the major yeast phospholipids (Fig. 2e), which is consistent with the ER expansion phenotype.

The unfolded protein response (UPR) has been shown to control the size of the ER under stress conditions^30,31. However, basal UPR levels in pex30∆fld1∆ or pex30∆ldb16∆ were comparable to WT cells, suggesting that ER expansion in these cells is UPR-independent (Supplementary Figure 3c). Expanded and convoluted ER membranes have also been observed during ER-phagy, a selective form of autophagy involved in ER turnover^32,33,34. However, abrogation of autophagy by deletion of the master regulator kinase Atg1 did not prevent formation of aberrant ER structures in pex30∆fld1∆ or pex30∆ldb16∆ cells (Supplementary Figure 3d). Moreover, in cells expressing the abundant ER marker Sec63-GFP, we did not detect accumulation of free green fluorescent protein (GFP) resulting from autophagic degradation of the ER^32,35 (Supplementary Figure 3e). Thus, the expanded and convoluted ER membranes in pex30∆fld1∆ or pex30∆ldb16∆ appear to originate independently of ER-phagy.

Pex30 was shown to have ER-shaping functions, an activity dependent on its reticulon-homology domain (RHD)⁵. Thus, we wondered whether disruption of major ER-shaping proteins³⁶, such as the reticulons Rtn1 and Yop1, would also result in defects similar to the ones detected in pex30∆fld1∆ and pex30∆ldb16∆ cells. Remarkably, combined mutations in seipin and the reticulons Rtn1 and Yop1 in rtn1∆yop1∆fld1∆ or rtn1∆yop1∆ldb16∆ cells did not result in Bodipy dispersal in ER membranes or near absence of LDs (Supplementary Figure 3f), suggesting the effects were specific to Pex30. Collectively, these data indicate that combined deletions of seipin and Pex30 block efficient LD budding resulting in trapping of neutral lipids in dramatically enlarged ER.

As a first step to dissect the relation between seipin and Pex30 in LD biogenesis, we analyzed their relative distribution. In WT cells growing in glucose-containing media, the vast majority of Fld1-tdTomato foci were proximal to LDs, at ER-LD contacts as previously reported^21,25. In contrast, only a small fraction of Pex30-mNG foci were proximal to LDs. Strikingly, under conditions favoring LD biogenesis, such as in oleic acid-containing media, both Fld1 and Pex30 were strongly concentrated at ER-LD contacts (Fig. 3). Thus, Fld1 appears to maintain a more stable association with ER-LD contacts, whereas Pex30 associates more transiently, perhaps only during earlier stages of LD biogenesis. Despite the partial colocalization between Pex30 and seipin, we failed to detect a biochemical interaction between these proteins (Supplementary Figure 4). This observation further supports the notion that seipin and Pex30 have partly redundant but independent roles in the budding of LDs from the ER.

Pex30 and seipin cooperate during peroxisome biogenesis

The seipin-Pex30 collaboration in LD budding prompted us to test whether these proteins also cooperate in peroxisome biogenesis, in which Pex30 was shown to have a role^5,6,26,37,38. To follow de novo peroxisome biogenesis, we used a well-established assay involving regulated expression of Pex3, a protein essential for formation of functional peroxisomes³⁹. Upon a GFP-Pex3 pulse, the import of a fluorescent reporter containing a peroxisomal targeting signal (mCherry-PTS1) from the cytoplasm into the matrix of newly formed peroxisomes can be monitored over time by microscopy (Fig. 4a). As previously reported⁵, pex30∆ mutants displayed a slight delay in peroxisome biogenesis. Mutations in the seipin complex components fld1∆ or ldb16∆ also resulted in a delay in peroxisome formation. Remarkably, pex30∆fld1∆ or pex30∆ldb16∆ mutants were strongly impaired in de novo peroxisome biogenesis without a single import-competent peroxisome forming during the time course of the experiments (Fig. 4b,c). The defect was not due to different Pex3 levels, as these were similar in all the strains analyzed (Supplementary Figure 5a). Aberrant peroxisome biogenesis in pex30∆fld1∆ and pex30∆ldb16∆ cells was also supported by analysis of mCherry-PTS1 at steady state (Fig. 4d). Although mCherry-labeled peroxisomes were detected in virtually every WT and single mutant cells, only 40% of pex30∆fld1∆ and pex30∆ldb16∆ cells contained peroxisomes (Fig. 4e). In fact, even cells with peroxisomes displayed a detectable cytoplasmic pool of mCherry-PTS1, indicative of compromised import of the matrix protein marker (Fig. 4d). Consistent with these defects, the prototypical peroxisomal membrane protein, Pex14, a component of the docking complex essential for matrix protein import⁴⁰, localized abnormally in pex30∆fld1∆ and pex30∆ldb16∆ cells. By fluorescence microscopy, we detected two to three foci of endogenously expressed Pex14-GFP per cell on each focal plane. In contrast, pex30∆fld1∆ and pex30∆ldb16∆ mutants displayed a diffused pattern with a few interspersed foci overlapping with Sec63-tdTomato, indicating its mislocalization to the ER (Fig. 4f). Thus, although mutations in seipin or Pex30 have mild defects in peroxisome formation, this process is strongly affected in double mutants, suggesting partial redundancy between seipin and Pex30 during peroxisome biogenesis.

Given the striking defects in peroxisomes and LDs, we wondered whether the formation of other ER-derived organelles, such as COPII vesicles would also be affected in cells lacking seipin and Pex30. Remarkably, the ER to Golgi trafficking of carboxypeptidase Y, a well-studied and abundant COPII cargo, was unaffected in pex30∆fld1∆ and pex30∆ldb16∆, indicating that the effects were specific to LDs and peroxisomes (Supplementary Figure 5b). Altogether, our data indicate that seipin and Pex30 have distinct but partly redundant roles specifically in the formation of LDs and peroxisomes. Moreover, it reveals previously unidentified links in the biogenesis of these structurally unrelated organelles.

Seipin is enriched at ER sites of PPV budding

During de novo peroxisome biogenesis, peroxisomal membrane proteins such as Pex3 and Pex14 are initially targeted to the ER, from where they leave in PPVs that eventually mature into peroxisomes^7,8,9,39. The budding of PPVs occurs at ER domains enriched in Pex30⁵. Thus, we asked whether seipin components also concentrate at the same ER domains by following de novo peroxisome biogenesis in cells expressing Fld1-tdTomato. Remarkably, at early time points upon induction, GFP-Pex3 almost invariably colocalized with Fld1-tdTomato (Fig. 4g, h). The degree of colocalization decreased over time and was negligible after 90 min (Fig. 4g, h), suggesting complete GFP-Pex3 incorporation into PPVs at this time point. Thus, similar to Pex30⁵, seipin is enriched at ER domains involved in PPV budding.

Loss of Pex30 and seipin leads to toxic TAG accumulation in ER membranes

So far, we showed that the biogenesis of LDs and peroxisomes from the ER depends on the cooperation of seipin and Pex30. Moreover, in their absence, peroxisomal membrane proteins such as Pex14 and TAG appear trapped in the ER. Strikingly, essential ER functions in protein trafficking and phospholipid biosynthesis do not appear to be greatly compromised in pex30∆fld1∆ and pex30∆ldb16∆ cells, and are unlikely to be responsible for the growth defect in these mutants. Although these mutants have only a slight increase in TAG (Fig. 5a), it is possible that in the absence of LDs, ER-trapped TAG becomes toxic. To test this hypothesis, we reduced TAG levels in pex30∆fld1∆ and pex30∆ldb16∆ cells by deleting the major TAG-synthesizing enzyme DGA1. Remarkably, in pex30∆fld1∆dga1∆ and pex30∆ldb16∆dga1∆ triple mutants, the growth defect was largely suppressed at all temperatures (Fig. 5b). Lipidomics analysis confirmed that DGA1 deletion indeed resulted in decreased TAG levels in the triple mutants (Fig. 5a). In addition, both the fraction of cells displaying Bodipy dispersal and the size of these structures were decreased in the triple mutants (Fig. 5c, d; Supplementary Figure 6a). In parallel, normal ER morphology was restored in a substantial fraction of cells (Fig. 5e, f), whereas PC and PI dropped to WT levels (Supplementary Figure 6b). Importantly, these changes resulted in a strong alleviation of the peroxisome biogenesis defect (Supplementary Figure 6c). Thus, TAG trapped in ER membranes is toxic and accounts for the slow growth of pex30∆fld1∆ and pex30∆ldb16∆ cells.

Seipin and Pex30 stabilize ER domains permissive for budding

Bilayer phospholipid composition and surface tension were recently identified as key factors for the budding of artificial LDs from model membranes in vitro⁴¹ and also appear to regulate LD budding in vivo⁴². Although major global lipidome changes were not detected in cells lacking seipin^4,20,25 or Pex30 (Supplementary Figure 7a), both proteins are capable of affecting membrane properties locally at sites of organelle biogenesis^5,22,43,44. Thus, an appealing mechanism for seipin and Pex30 to function is through organization of ER domains with lipid composition permissive for organelle budding and that are distinct from bulk ER membranes. In this case, in pex30∆fld1∆ and pex30∆ldb16∆ cells the budding of LDs and PPVs would be dependent on the bulk phospholipid composition of the ER. Indeed, pex30∆fld1∆ and pex30∆ldb16∆ cells have high levels of diacylglycerol (Fig. 6a), a lipid that disfavors positive membrane curvature and strongly inhibits LD budding^41,42. A second prediction of the model is that conditions generating a more budding-permissive lipid composition should restore organelle budding in pex30∆fld1∆ and pex30∆ldb16∆ mutants. Remarkably, the formation of both LDs (Fig. 6b and Supplementary Figure 7b) and peroxisomes (Fig. 6c, d and Supplementary Figure 7c) were restored in the absence of seipin and Pex30 upon deletion of Pct1, a rate-limiting enzyme in the Kennedy pathway of phospholipid biosynthesis. In pex30∆fld1∆pct1∆ and pex30∆ldb16∆pct1∆ mutants, the presence of LDs resulted in a concomitant decrease of cells with dispersed Bodipy structures again, suggesting that these correspond to ER-trapped TAG (Supplementary Figure 7d). It should be noted that LDs in these cells were supersized, similar to those detected in seipin mutants^20,21,25, indicating that besides budding, seipin controls other aspects of LD biogenesis. In these triple mutants, besides lower PC levels, which were expected due to deletion of PCT1^45,46, there was a reduction in PI (Fig. 6e) and DAG (Fig. 6a) to levels comparable to WT cells. Concomitantly, there was a strong increase in TAG levels (Fig. 6f). Thus, in the absence of seipin and Pex30, budding of LDs and PPVs is entirely dependent on ER phospholipid composition.

Discussion

LDs and peroxisomes are central organelles in cellular metabolism and, in a variety of cell types, are often in close proximity^47,48,49. However, the mechanisms by which peroxisomes and LDs form at the ER remain elusive. Here we present evidence that PPVs, which originate peroxisomes, and LDs bud from similar ER domains through a process requiring the cooperation between seipin and Pex30. Simultaneous deletion of these components largely inhibits budding of LDs and PPVs, indicating their partly redundant roles in organelle biogenesis. Remarkably, remodeling of ER phospholipid composition restores organelle budding in the absence of seipin and Pex30, highlighting the role of ER membrane composition in organelle biogenesis.

Curiously, neither seipin nor Pex30 appear to modify lipids directly. In fact, individual deletions of seipin or PEX30 do not result in major global changes in lipid composition^20,22,50. Thus, it was puzzling that simultaneous ablation of seipin and Pex30 resulted in dramatic lipidome remodeling, with a strong increase in PC and PI, and to a lower extent, DAG levels. A reduction in TAG synthesis by deletion of DGA1 largely corrected phospholipid levels in these cells indicating that the defects resulted from inhibition of LD budding and consequent TAG dispersal through ER membranes. Perhaps when trapped in the ER, TAG is more susceptible to lipolysis, resulting in DAG, which accumulates in the ER, as well as in free fatty acids likely shunted to phospholipid synthesis. According to recent in vitro and in vivo studies, DAG is a potent inhibitor of LD budding^41,42. Given the link between LD and PPV biogenesis uncovered here, we speculate that high amounts of DAG in the ER may also inhibit PPV budding. Consistent with this scenario, deletion of DGA1 which lowers DAG levels, restores peroxisome formation in the absence of seipin and Pex30.

The abnormal lipid profile of seipin∆pex30∆ mutants was also corrected by deletion of PCT1, a rate-limiting enzyme of the Kennedy pathway, in which DAG and choline are used to synthesize PC⁵¹. Thus, a decrease in PC levels was expected in cells lacking Pct1. In contrast, the reduction in DAG is paradoxical as a block in the Kennedy pathway would prevent its consumption. However, concomitant with the reduction of DAG, seipin∆pex30∆pct1∆ cells display a strong increase in TAG levels. Thus, by lowering DAG levels and relieving the inhibitory effect of this lipid in organelle budding^41,42, PCT1 deletion facilitates TAG storage in LDs in seipin∆pex30∆ cells. Similarly, the reduction in DAG probably also accounts for the restoration of peroxisome formation in seipin∆pex30∆pct1∆ cells. We previously showed that Pct1 relocalizes strongly to LDs in seipin mutants²², a phenotype also observed in seipin-deficient Drosophila cultured cells⁴. Thus, regulation of Pct1 accumulation and/or activity may be one of the seipin functions at sites of organelle budding.

Pex30 has a RHD with membrane-shaping properties⁵ that is required for normal peroxisome biogenesis^6,26,37,38. Moreover, in its C-terminal cytosolic domain, Pex30 contains a dysferlin-like domain that may bind DAG^38,52,53. Whether both membrane-shaping and DAG-binding activities are necessary to promote organelle budding in seipin mutants is unclear and should be addressed in the future. However, an appealing possibility is that Pex30 may regulate DAG distribution locally, at sites of organelle biogenesis, an activity that may become essential in the absence of seipin or unregulated Pct1 activity.

Pex30 was also shown to interact with Pex28, Pex29, Pex31 and Pex32, as well as Rtn1 and Yop1^6,37. The significance of these interactions is not yet clear but it is interesting to note that all these proteins have membrane-shaping properties. However, Pex30-interacting factors do not appear to display genetic interactions with seipin (Supplementary Figure 2 and our unpublished results). Thus, Pex30-mediated organelle budding appears independent of its canonical binding partners. Moreover, ER organelle budding still occurred in cells with combined deletions in seipin and the major ER-shaping proteins Rtn1 and Yop1, suggesting that the defects in seipin∆pex30∆ cells are specific and not due to global defects in ER morphology.

How seipin and Pex30 promote organelle biogenesis is not fully understood, but our data supports a model in which these components organize ER domains with a lipid composition distinct from the bulk ER that is permissive for organelle budding. Peroxisomes and LDs are structurally distinct so it will be important to understand how seemingly similar ER sites give rise to monolayered LDs and bilayered PPVs. Despite their differences, both organelles are intimately linked to cellular metabolism and are made in response to similar stimuli³. Thus, it is appealing to speculate that the similar requirements during their biogenesis may reflect a common evolutionary origin for these organelles.

Methods

Reagents

GFP antibody (rat monoclonal 3H9; dilution 1:1000) was purchased from Chromotek and PGK1 antibody (mouse monoclonal 459250; dilution 1:10,000) from Invitrogen. Anti-CPY (rabbit polyclonal ab34636; dilution 1:750) was purchased from Abcam. Polyclonal anti-Fld1 (rabbit; dilution 1:3000), anti-Ldb16 (rabbit; dilution 1:5000), anti-Dga1 (rabbit; dilution 1:3000), and anti-Ldo (rabbit; dilution 1:3000) antibodies were previously described^22,54. Pex30 antibody (rabbit; dilution 1:1000) was a kind gift from William Prinz (National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, USA). LD dyes Bodipy493/503 was purchased from Invitrogen and used at a final concentration of 1 µg/ml. Monodansyl pentane (MDH) was purchased from Abgent and used at 0.1 mM.

Yeast strains and plasmids

Endogenous protein tagging and promoter replacement were performed using standard PCR-based homologous recombination as described^55,56. Deletion mutants were made either by PCR-based homologous recombination or by crossing haploid cells of opposite mating types, followed by sporulation and tetrad dissection using standard protocols⁵⁷. Strains used are isogenic to either BY4741 (MATa ura3∆0 his3∆1 leu2∆0 met15∆0) or BY4742(MATα ura3∆0 his3∆1 leu2∆0 lys2∆0), and are listed in Table S3.

Growth conditions

Cells were grown at 30 °C in YPD liquid medium (1% yeast extract, 2% peptone, 2% glucose) unless indicated otherwise. For experiments using oleic acid, cells were grown to diauxic shift phase in YPD, pelleted, and resuspended in YP + 0.1% oleic acid. For microscopy experiments, exponentially growing cells were analyzed. For growth assays, cells were grown to exponential phase in the indicated media (either YPD or SC), diluted to an OD₆₀₀ of 0.1 followed by 10-fold serial dilutions. Diluted cells were spotted on either YPD or synthetic complete (SC) plates, and incubated at the indicated temperatures for 2 days.

De novo peroxisome biogenesis

De novo peroxisome biogenesis was analyzed as previously described³⁹. In brief, cells were grown in a starter culture in YPD, diluted in YP + 4% raffinose, and grown to exponential phase. At this stage, cells were washed and resuspended in YP + 2% galactose. After a 30 min galactose pulse to induce expression of GFP-Pex3, cells were washed, resuspended in YP + 4% glucose, and analyzed at the indicated time points.

Immunoprecipitation

Immunoprecipitation of endogenously tagged Fld1-TAP and Ldb16-TAP was performed as previously described⁵⁴. Briefly, 100 OD₆₀₀ units of yeast culture in log phase were washed and resuspended in 1.4 ml lysis buffer (LB; 50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM MgCl₂, and cOmplete protease inhibitor [Roche]). Cells were lysed with glass beads and lysates were cleared by low-speed centrifugation at 4 °C. Membranes were pelleted at 135,000 × g for 25 min at 4 °C in an Optima Max Tabletop Ultracentrifuge in a TLA 100.3 rotor (Beckman Coulter). The crude membrane fraction was resuspended in 600 μl LB. 700 μl of LB supplemented with digitonin to obtain 1% final concentration and membranes were solubilized for 2–3 h on a rotating wheel at 4 °C. Solubilized membranes were cleared for 15 min at 4 °C at full speed in a tabletop centrifuge, and 1.1 ml was used for the immunoprecipitation. TAP -tagged proteins were affinity isolated by overnight incubation with calmodulin sepharose 4B beads (17052901; GE Healthcare). Eluted proteins were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting. In all experiments, the input lane corresponds to 5% of the total extract used for immunoprecipitation.

Pulse-chase and protein analysis

For pulse-chase analysis, 9 OD units of exponentially growing cells starved for methionine and cysteine were pulse-labeled for 5 min with 250 μCi of [³⁵S] methionine/cysteine followed by addition of cold methionine/cysteine to a final concentration of 2 mM. Collected time points were lysed and CPY was immunoprecipitated with anti-CPY antibody for 2 h at 4 °C. Subsequently, immunocomplexes were retrieved by an additional 1 h incubation at 4 °C with Protein A Sepharose beads. Eluted proteins were separated by SDS-PAGE and visualized by autoradiography. For western blotting, whole-cell extracts of exponentially growing cells were prepared from 2 OD units of cells. Pelleted cells were washed once in phosphate-buffered saline, resuspended in 250 µL of 0.15 M NaOH, and incubated on ice for 10 min. After centrifugation at maximum speed for 2 min at 4 °C, the pellet was resuspended in 1 × sample buffer and heated at 65 °C for 10 min. Proteins were separated by SDS-PAGE in Criterion TGX™ pre-casted gels (BioRad), transferred to PVDF membrane, and analyzed with the indicated antibodies.

Fluorescence microscopy

Most experiments were performed in a Zeiss Cell Observer HS equipped with a CMOS camera (Hamamatsu ORCA-Flash4.0) controlled by 3i Slidebook6.0 software. A Plan-APOCHROMAT 100 × 1.4 objective was used. GFP and BODIPY493/503, mCherry, and MDH signals were detected using GFP, RFP and DAPI filters, respectively, with standard settings. Analysis of ER morphology in Sec63-mNG expressing cells was performed in a Zeiss 880 Airyscan microscope using a 63 × 1.4 Plan-APOCHROMAT objective. All imaging were performed at room temperature.

Image analysis

Analysis of dispersed Bodipy structures was performed using 3iSlidebook 6.0 software. Masks were created manually for each dispersed Bodipy structure observed on a single z-position, and volume (in µ³) was measured. Statistical significance and P-values were calculated in GraphPad Prism 7 using the Mann–Whitney test.

Electron microscopy

Exponentially growing cells were pelleted, loaded into 3 mm, 100 µm, or 200 µm carriers and high-pressure frozen using a Leica EM ICE. Samples were freeze substituted in a Leica AFS2 unit in 1% uranyl acetate +1% water in acetone. Samples were then maintained at − 45 °C for the remainder of the processing. Samples were washed with acetone and then ethanol for 15 min each, then infiltrated with HM20 resin (PolySciences) according to the following schedule: 25% resin in ethanol for 4 h, 50% resin in ethanol overnight, 75% resin in ethanol for 5 h, 100% resin for 6 h, 100% resin overnight, 100% resin for 8 h. Samples were then polymerized with UV light for 48 h, and gradually warmed to 0 °C after the first 24 h of polymerization. 90 nm ultranthin sections were acquired using a Diatome diamond knife with a Leica UC7 ultramicrotome and transferred to formvar-coated 100-mesh copper grids. Sections were post-stained for 10 min with 2% uranyl acetate, washed by passing over a series of warm water droplets, and then stained with Reynold’s lead citrate, washed, and dried. Grids were imaged in a FEI Tecnai 12 TEM operated at 120 kV using a Gatan OneView digital camera.

LD isolation

LD purification was carried out as described²². Briefly, cells were grown in YPD until stationary phase. Cells (3000 ODs) were centrifuged at 3,000 × g for 5 min (J J26-XP centrifuge and JLA8100 rotor; Beckman Coulter), washed in milliQ water, preincubated in 0.1 M Tris-HCl, pH9.5, and 10 mM dithiothreitol for 10 min at 30 °C, washed, and resuspended in 50 mL spheroplasting buffer (1.2 M sorbitol and 50 mM Tris, pH 7.4). To prepare spheroplasts, Zymolyase 20T (Seikagaku Biobusiness) was added (10 µg/OD₆₀₀ U cells) followed by incubation in a water bath at 30 °C until a 10-fold drop in OD₆₀₀ was observed (45–60 min). Spheroplasts were recovered by centrifuging at 1,000 × g at 4 °C, washed with spheroplasting buffer, and resuspended in breaking buffer (BB: 10 mM MES, Tris, pH 6.9, 12 % [wt/wt] Ficoll 400, 0.2 mM EDTA, 1 mM phenylmethane sulfonyl fluoride and protease inhibitor cocktail). Cells were homogenized using a Dounce homogenizer on ice, and the homogenate centrifuged (5000 × g for 5 min) using rotor JS13.1. The resulting supernatant was centrifuged for 45 min at 175,000 × g (Optima L-100K centrifuge, Beckman Coulter; SW-32 rotor) and the LD-containing floating layer resuspended in BB, overlaid with 19 mL 10 mM MES-Tris, pH 6.9, 8% [wt/wt] Ficoll 400, and 0.2 mM EDTA, and centrifuged as before. The floating layer was collected and resuspended in 10 mM MES-Tris, pH 6.9, 8% [wt/wt] Ficoll 400, 0.6 M sorbitol, and 0.2 mM EDTA, overlaid with 19 mL 10 mM MES-Tris, pH 6.9, 0.25 M sorbitol, and 0.2 mM EDTA, and centrifuged once more at 175,000 × g for 30 min. The highly purified top LD fraction was snap frozen, stored at −80 °C, and used subsequently for proteomics.

Protein mass spectrometry

Label-free proteome quantification was carried out as described²². Briefly, trichloroacetic acid precipitated proteins were resuspended in 6 M urea and 200 mM ammonium bicarbonate before reduction (10 mM dithiothreitol) and alkylation (20 mM iodoacetamide). Samples were diluted to 2 M urea and digested with trypsin (1:10 wt/wt) overnight at 37 °C. Tryptic peptide mixtures were desalted using a C18 UltraMicroSpin column using three washes with 0.1% formic acid in water, followed by an elution step with 0.1% formic acid in a 1:1 mix of water and acetonitrile⁵⁸. Samples were analyzed in a LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific) coupled to nano-LC (Proxeon) equipped with a reversed-phase chromatography 12 cm column with an inner diameter of 75 µm, packed with 5 µm C18 particles (Nikkyo Technos). Chromatographic gradients were set from 93% buffer A, 7% buffer B to 65% buffer A, 35% buffer B in 60 min with a flow rate of 300 nl/min (buffer A: 0.1% formic acid in water; buffer B: 0.1% formic acid in acetonitrile). The instrument was operated in DDA mode and full mass spectrometry scans with 1 micro scans at resolution of 60,000 were used over a mass range of m/z 250–2,000 with detection in the Orbitrap. After each survey scan, the top 20 most intense ions with multiple charged ions above a threshold ion count of 5000 were selected for fragmentation at normalized collision energy of 35%. Fragment ion spectra produced via collision-induced dissociation were acquired in the linear ion trap. All data were acquired with Xcalibur software v2.2. Acquired data were analyzed using the Proteome Discoverer software suite (v1.3.0.339; Thermo Fisher Scientific), and the Mascot search engine (v2.3; Matrix Science) was used for peptide identification. Data were searched against database containing all yeast proteins according to the Saccharomyces Genome Database plus the most common contaminants⁵⁹. A precursor ion mass tolerance of 7 ppm at the MS1 level was used, and up to three miscleavages for trypsin were allowed. The fragment ion mass tolerance was set to 0.5 D. Oxidation of methionine and protein acetylation at the N-terminus were defined as variable modifications. Carbamidomethylation on cysteines was set as a fix modification. The identified peptides were filtered using a false discovery rate < 5%. Protein areas were normalized intra- and intersamples by median of the log area. A linear modeling approach implemented in lmFit function and the empirical Bayes statistics implemented in eBayes and topTable functions of the Bioconductor limma package^60,61 were used to perform a differential protein abundance analysis. The normalized protein areas of different yeast mutants were compared with wt samples. Protein p-values were calculated with limma and were adjusted with Benjamini–Hochberg method⁶². A value of 0.05 was used as a cutoff.

Mass spectrometric lipid analysis

For lipidome analysis cells were grown in YPD until OD₆₀₀ = 2. Lipid extraction and mass spectrometry analysis were done as described previously^28,63. Briefly, yeast cells were resuspended in 150 nM NH₄HCO₃ (pH 8) and disrupted by zirconia beads (0.5 mm; Biospec Products). Cell lysates were diluted to 0.2 OD units per 200 µL, and mixed with 20 µL of a cocktail of 20 internal lipid standards. Samples were extracted with 990 μL chloroform/methanol (17:1, V/V) for 120 min. The lower organic 17:1 phase lipid extract was collected. The remaining aqueous sample material was re-extracted with 990 μL chloroform/methanol (2:1, V/V) for 120 min. The lower organic 2:1 phase lipid extract was collected. The lipid extracts were vacuum evaporated. Finally, the lipid extracts were dissolved in 100 μL chloroform/methanol (1:2, V/V). Lipid extracts were analyzed in negative and positive ion mode on a QSTAR Pulsar-i instrument (MDS Analytical Technologies) and a LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific) both equipped with the robotic nanoflow ion source TriVersa NanoMate (Advion Biosciences) as described^64,65,66. Detected lipid species were identified and quantified using Lipid Profiler (MDS Analytical Technologies) and proprietary Analysis of Lipid EXperiments (ALEX) software.

Statistical analysis

Data for all microscopy experiments were generated from at least two independent experiments. For each experiment, 150 cells were scored per condition and/or genotype from multiple microscopy fields. Distributions are presented as mean ± SD. Where indicated, the volume of dispersed bodipy structures was measured using the 3iSlidebook 6.0 software and statistical significance tested using the Mann–Whitney test in GraphPad Prism 7. Two-tailed P-values are indicated in figures where relevant. Proteomics data result from analysis of six (for WT and ldb16∆) and two (for fld1∆) independent LD isolation experiments. Lipidomics data result from two independent biological replicates for all genotypes, each analyzed in two independent technical replicates. In all experiments, cells were randomly selected for analysis and no samples were excluded.

Data availability

The data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

Mass spectrometric protein measurements and data analysis were performed in the Centre for Genomic Regulation (CRG)/Universitat Pompeu Fabra Proteomics Unit, part of the “Plataforma de Recursos Biomoleculares y Bioinformàticos (ProteoRed-Instituto de Salud Carlos III, PT13/0001)”. EM samples were prepared at the Dunn School EM Facility with the help of E. Johnson. We thank Sebastian Schuck for reagents, W. Prinz for sharing results before publication, and F. Campelo and the Carvalho Lab for discussions and critical reading of the manuscript. C.S.E. is supported by the VILLUM Foundation (VKR023439) and the VILLUM Center for Bioanalytical Sciences (VKR023179). P.C. is supported by an ERC starting grant, a Wellcome Trust Investigator Award and the EMBO Young Investigator Program.

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These authors contributed equally: Sihui Wang, Fatima-Zahra Idrissi.

Authors and Affiliations

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK
Sihui Wang & Pedro Carvalho
Cell and Developmental Biology Programme, Centre for Genomic Regulation (CRG), Dr. Aiguader, 88, 08003, Barcelona, Spain
Fatima-Zahra Idrissi, Alexandra Grippa & Pedro Carvalho
Universitat Pompeu Fabra (UPF), Dr. Aiguader, 88, 08003, Barcelona, Spain
Fatima-Zahra Idrissi, Alexandra Grippa & Pedro Carvalho
Department of Biochemistry and Molecular Biology, Villum Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, 5230, Odense, Denmark
Martin Hermansson & Christer S. Ejsing
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany
Christer S. Ejsing

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Sihui Wang
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Fatima-Zahra Idrissi
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Martin Hermansson
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Alexandra Grippa
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Contributions

:S.W. and F.I. performed most of the experiments and data analysis. M.H. performed lipid extractions, lipid mass spectrometry. and lipidomics data processing with the supervision of C.S.E. A.G. prepared LD-enriched fractions for quantitative proteomics. P.C. conceived and guided the project, analyzed the data, and wrote the manuscript with input from all the authors.

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Correspondence to Pedro Carvalho.

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Wang, S., Idrissi, FZ., Hermansson, M. et al. Seipin and the membrane-shaping protein Pex30 cooperate in organelle budding from the endoplasmic reticulum. Nat Commun 9, 2939 (2018). https://doi.org/10.1038/s41467-018-05278-2

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Received: 08 September 2017
Accepted: 18 June 2018
Published: 27 July 2018
DOI: https://doi.org/10.1038/s41467-018-05278-2

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