Fibroblast Growth Factors (FGFs) form a family of more than 20 potent mitogens that stimulate the growth of a wide range of cells including fibroblasts and endothelial cells (Bikfalvi et al., 1997). They are of critical importance for physiological processes such as embryonic development, tissue regeneration, wound repair and hematopoiesis (Bikfalvi et al., 1997). FGF2 is the prototype member of this family that, beyond the functions of FGFs in normal cell growth and differentiation, plays critical roles under pathophysiological conditions (Akl et al., 2016). This is particularly evident in the context of cancer with FGF2 being a major mediator of tumor-induced angiogenesis (Presta et al., 2005). In addition, FGF2 acts as a survival factor that inhibits tumor cell apoptosis by an autocrine secretion-signaling loop (Noh et al., 2014; Pardo et al., 2006). This process is believed to represent a frequent cause of tumor cell resistance against conventional anti-cancer therapies.

As opposed to other FGF family members, FGF2 lacks a signal peptide and is transported into the extracellular space by an ER/Golgi-independent mechanism (La Venuta et al., 2015; Nickel, 2011; Nickel and Rabouille, 2009, 2008). The term unconventional secretion has been used for four different pathways of protein transport towards the plasma membrane and the extracellular space (Rabouille et al., 2012). Two of these pathways concern soluble proteins derived from the cytoplasm that are transported into the extracellular space by either direct protein translocation across the plasma membrane (type I unconventional secretion) or vesicular mechanisms involving endosomal compartments (type III unconventional secretion) (Rabouille et al., 2012; Zhang and Schekman, 2013; Piccioli and Rubartelli, 2013). Based on compelling evidence from both cell-based FGF2 secretion experiments and biochemical in vitro studies, unconventional secretion of FGF2 was shown to follow a type I mechanism that is based on direct protein translocation across the plasma membrane (La Venuta et al., 2015; Zhang and Schekman, 2013; Malhotra, 2013; Nickel, 2005; Schäfer et al., 2004). This process depends on sequential interactions of FGF2 with the phosphoinositide PI(4,5)P₂ at the inner leaflet and heparan sulfate proteoglycans at the outer leaflet of the plasma membrane (Nickel, 2011; Nickel and Rabouille, 2009; Nickel, 2007; Temmerman et al., 2008; Zehe et al., 2006). Recruitment by PI(4,5)P₂ triggers oligomerization of FGF2 at the inner leaflet resulting in membrane insertion, a key step in FGF2 membrane translocation (La Venuta et al., 2015; Steringer et al., 2012, 2015). The membrane inserted state of FGF2 oligomers has been proposed to be linked to a membrane pore with a toroidal architecture (Steringer et al., 2012). This view is supported by the observation that membrane lipids undergo transbilayer diffusion (Steringer et al., 2012). It appears possible that PI(4,5)P₂ molecules themselves redistribute between monolayers when FGF2 oligomers insert into the membrane as it has been reported in other systems (Bucki et al., 2000). Membrane insertion of FGF2 oligomers is stimulated by phosphorylation of tyrosine 81 in FGF2 mediated by the non-receptor tyrosine kinase Tec, a trans-acting factor that is associated with the inner leaflet through an interaction of its PH domain with PI(3,4,5)P₃ (Steringer et al., 2012; Ebert et al., 2010; La Venuta et al., 2016). Recently, the integral membrane protein ATP1A1 has been identified as another trans-acting factor in unconventional secretion of FGF2 (La Venuta et al., 2015; Zacherl et al., 2015) FGF2 was shown to directly interact with the cytoplasmic domain of ATP1A1 (La Venuta et al., 2015; Zacherl et al., 2015), however, the precise function of ATP1A1 in FGF2 secretion is unknown. In addition to the cis elements that are important for FGF2 binding to PI(4,5)P₂ (K127/R128/K133) and heparan sulfates (K133) as well as Y81 being the target of Tec kinase, membrane insertion of FGF2 oligomers depends on two cysteine residues (C77/C95) on the molecular surface of FGF2 (La Venuta et al., 2015; Müller et al., 2015). Intriguingly, despite a high degree of overall amino acid conservation among FGFs, C77 and C95 are absent from all members of the FGF family carrying signal peptides. C77/C95 have been shown to form intermolecular disulfide bridges, a process that drives efficient oligomerization of FGF2 in a PI(4,5)P₂-dependent manner (Müller et al., 2015). Following membrane insertion, cell surface heparan sulfate proteoglycans are required to complete membrane translocation by disassembling FGF2 oligomers at the outer leaflet and trapping of FGF2 in the extracellular space (Nickel, 2007; Zehe et al., 2006; Seelenmeyer et al., 2005). The interaction between FGF2 and cell surface heparan sulfates is mediated by basic residues in the C-terminal part of FGF2 with K133 being an essential component of this binding motif (Nickel and Rabouille, 2009, 2008; Temmerman et al., 2008). Thus, four cis-elements in FGF2 [K127/R128/K133 forming the PI(4,5)P₂ binding pocket and K133 involved in FGF2 binding to both PI(4,5)P₂ and heparan sulfates, Y81 being the target of Tec kinase and C77/C95 promoting FGF2 oligomerization] and four trans-acting factors [PI(4,5)P₂, ATP1A1, Tec kinase and heparan sulfate proteoglycans] have been identified to participate in unconventional secretion of FGF2 from cells. All known trans-acting factors are physically associated with the plasma membrane, the subcellular site of membrane translocation during unconventional secretion of FGF2 (La Venuta et al., 2015).

The molecular mechanism by which FGF2 is secreted from cells might be similar to other proteins secreted by unconventional means. This includes HIV-Tat that has been demonstrated to be secreted from infected T cells in a PI(4,5)P₂ dependent manner (Debaisieux et al., 2012; Rayne et al., 2010a, 2010b). Consistently, HIV-Tat was recently found to bind to PI(4,5)P₂ concomitant with the formation of membrane pores (Zeitler et al., 2015). Interestingly, a recent study suggested a role for membrane pores in unconventional secretion of Interleukin 1β from macrophages (Martín-Sánchez et al., 2016). While Interleukin 1β on its own cannot form pores in the plasma membrane, its release from macrophages might be coupled to another factor that becomes activated in an inflammasome-dependent manner, gasdermin (Ding et al., 2016). Since gasdermin is capable of forming membrane pores in a PI(4,5)P₂ dependent manner, it appears possible that at least one pathway of Interleukin 1β secretion exists that is based upon membrane pores that are formed by gasdermin oligomers upon activation of inflammasomes. Therefore, the molecular mechanisms of unconventional secretion of FGF2, HIV-Tat and even Interleukin 1β might be related in mechanistic terms.

In the current study, we tested key predictions of the FGF2 membrane translocation model as well as investigated the structure-function relationship of membrane-inserted FGF2 oligomers as intermediates in this process. Furthermore, we used atomistic molecular dynamics simulations to shed light on the initial steps of PI(4,5)P₂ triggered FGF2 oligomerization and membrane insertion. Using both biochemical and structural approaches, we demonstrate binding to FGF2 of PI(4,5)P₂ versus heparin to be mutually exclusive, a key aspect of the vectorial FGF2 membrane translocation model with heparan sulfates forming a molecular trap for FGF2 on cell surfaces. Furthermore, using giant unilamellar vesicles (GUVs) as a model system for the plasma membrane, we define the minimal molecular machinery required for FGF2 membrane translocation in a fully reconstituted inside-out system. These studies revealed only two trans-acting factors to be essential for FGF2 membrane translocation, PI(4,5)P₂ on GUV surfaces and long-chain heparins in the lumen of GUVs, the latter being used as mimetics of cell surface heparan sulfate proteoglycans. Consistently, the corresponding cis-elements required for FGF2 binding to PI(4,5)P₂ and heparan sulfates as well as the two cysteine residues required for oligomerization and membrane insertion of FGF2 were found essential for FGF2 membrane translocation. In addition, using various kinds of single molecule techniques, we demonstrate membrane-inserted forms of FGF2 to represent highly dynamic oligomers with a subunit number in the range of 8–12 FGF2 molecules. These studies were complemented by molecular dynamics simulations gaining the first insights into the initial steps of PI(4,5)P₂ induced oligomerization of FGF2. Most importantly, through simultaneous interactions of FGF2 monomers with multiple PI(4,5)P₂ molecules, a high affinity orientation of FGF2 was identified that favors FGF2 dimerization through a C95-C95 disulfide bridge along with additional electrostatic interactions within the dimerization interface. Our combined findings establish both the molecular basis and the minimal molecular machinery required for unconventional secretion of FGF2 from cells that consists of a surprisingly simple set of factors. Our findings further demonstrate the core mechanism of FGF2 membrane translocation to be thermodynamically driven by PI(4,5)P₂ dependent FGF2 oligomerization and membrane insertion without a requirement for additional energy sources such as ATP. The combination of PI(4,5)P₂ dependent FGF2 oligomerization, membrane insertion and heparan sulfate mediated trapping establishes a new type of self-sustained protein translocation across membranes that explains the molecular basis of how FGF2 is secreted from cells.

The current study is the first of its kind in which an unconventional mechanism of protein secretion has been reconstituted from purified components establishing the molecular mechanism by which FGF2 is secreted from cells. We define the minimal machinery required for FGF2 membrane translocation and provide novel insights into the structure function relationship of membrane inserted FGF2 oligomers, the key intermediates of this process. Finally, using atomistic molecular dynamics simulations, this study puts forward a mechanism by which FGF2 monomers assemble into dimers and trimers on membrane surfaces in a PI(4,5)P₂ dependent manner, the rate limiting step for the formation of higher oligomers that form membrane pores.

The first part of this work provides direct proof for two critical predictions of a previously proposed model describing the molecular mechanism of unconventional secretion of FGF2 from cells (La Venuta et al., 2015; Nickel, 2011; Nickel and Rabouille, 2009, 2008). The first predicted binding of FGF2 to PI(4,5)P₂ versus cell surface heparan sulfates to be mutually exclusive to ensure directional translocation of FGF2 across the plasma membrane based on sequential interactions of FGF2 with PI(4,5)P₂ at the inner leaflet and heparan sulfates at the outer leaflet. Here, using NMR spectroscopy we demonstrate an overlap of the binding sites on the molecular surface of FGF2 for IP₃ [the headgroup of PI(4,5)P₂] and a defined low affinity heparin disaccharide. In addition, we provide direct biochemical proof that long-chain heparins (used as mimetics of cell surface heparan sulfates) directly compete with membrane incorporated PI(4,5)P₂ for the interaction with FGF2. Based upon the high affinity of FGF2 towards heparan sulfates and long-chain heparins [K_D ≈ 100 nM (Faham et al., 1996)] and the lower affinity towards PI(4,5)P₂ in the low micromolar range (Temmerman et al., 2008; Temmerman and Nickel, 2009) (Figure 1), our findings provide a direct explanation for vectorial translocation of FGF2 from the cytoplasm to the cell surface.

The second prediction of the FGF2 secretion model proposed the minimal machinery required for FGF2 membrane translocation to be composed of two trans-acting factors, PI(4,5)P₂ and cell surface heparan sulfates. This in turn suggests a direct requirement for cis elements in FGF2 that mediate binding to PI(4,5)P₂ (K127/R128) and heparan sulfates (K133). In addition, the core mechanism involves FGF2 oligomerization and membrane pore formation, a process that depends on two cysteine residues on the molecular surface of FGF2 (C77/C95). Using GUVs with a plasma membrane like lipid composition along with a FGF2-GFP fusion protein, we demonstrate FGF2 membrane translocation into the lumen of GUVs to depend on the presence of PI(4,5)P₂ on membrane surfaces and the presence of long-chain heparins in the lumen of GUVs. Beyond these trans-acting factors, we demonstrate cis-elements in FGF2 required for binding to PI(4,5)P₂ and heparin as well as for FGF2 oligomerization and membrane pore formation to be essential for FGF2 membrane translocation in a fully reconstituted system. By contrast, the trans-acting factor ATP1A1, even though required for FGF2 secretion from cells (La Venuta et al., 2015; Zacherl et al., 2015), apparently is dispensable for FGF2 membrane translocation under the conditions used in this study. This indicates that ATP1A1 has regulatory functions in FGF2 secretion from cells rather than belonging to the core machinery mediating physical translocation of FGF2 across the plasma membrane.

In the second part of this study, we used single molecule techniques to gain insight into the structure function relationship of membrane inserted FGF2 oligomers as key intermediates in membrane translocation. First, we used supported lipid bilayers (SLBs) and single molecule confocal microscopy to determine the oligomeric size distribution of membrane-inserted FGF2 species. Following short incubation times of 10 min during which the overall integrity of SLBs was fully maintained, three major oligomeric species could be detected represented by trimers, hexamers and higher oligomers in the range of about 10 to 12 subunits. These findings were corroborated by an independent single molecule system in which the average oligomeric state of FGF2 could be correlated with membrane insertion and pore formation in GUVs. Here, even after incubation times of several hours at high concentrations of FGF2, the average oligomeric state of FGF2 in GUVs leveled out in the range of 8 to 12 subunits. On the one hand, these experiments demonstrate membrane-inserted FGF2 oligomers to represent highly dynamic structures that are not characterized by a uniform number of subunits. On the other hand, our findings demonstrate that PI(4,5)P₂ dependent membrane recruitment does not result in the random formation of undefined FGF2 aggregates. Therefore, as discussed above, we propose that membrane inserted FGF2 oligomers serve as dynamic translocation intermediates by controlled assembly through PI(4,5)P₂ dependent FGF2 oligomerization at the inner leaflet and controlled disassembly at the outer leaflet mediated by heparan sulfates. Previous evidence suggests that membrane inserted FGF2 oligomers are accommodated within a lipidic membrane pore with a toroidal architecture (La Venuta et al., 2015; Steringer et al., 2012). We propose that these translocation intermediates represent dynamic structures to which FGF2 subunits are constantly added at the cytoplasmic leaflet while FGF2 subunits are continuously removed at the extracellular side of the plasma membrane resulting in FGF2 translocation to the cell surface. It is currently unknown whether these assembly/disassembly units are monomers or disulfide bridged dimers of FGF2. Furthermore, it will be a challenge for future studies to elucidate the three-dimensional architecture of membrane inserted FGF2 oligomers to fully understand how these complexes are functioning as translocation intermediates in unconventional secretion of FGF2.

The third part of this study was concerned with the analysis of the initial events of FGF2 membrane translocation with a particular focus on the molecular mechanism of FGF2 interactions with PI(4,5)P₂ and the formation of dimers and trimers that initiate oligomerization of FGF2 in the context of PI(4,5)P₂ containing membranes. Atomistic simulations revealed the key binding site residues that interact with PI(4,5)P₂, which were found consistent with previous mutational analyses of FGF2 (6,17,19,25,33). Interestingly, the simulations predicted that there are additional previously unknown residues involved in membrane binding when several PI(4,5)P₂ head groups bind to FGF2 simultaneously. Therefore, the strength of FGF2 membrane interactions obviously depends on the number of PI(4,5)P₂ molecules that simultaenously bind to FGF2. In addition, the simulations provided compelling evidence that the strength of FGF2 membrane interactions also depends on the orientation of FGF2 relative to the membrane. The identified high-affinity orientation of FGF2 indeed favors multiple interactions of FGF2 with several PI(4,5)P₂ head groups and also leads to the formation of stable FGF2 dimers that are characterized by C95 – C95 disulfide bridges and additional ion pair interactions between residues within the dimerization interface. By contrast, the low-affinity orientation of FGF2 tilts the protein in a way that impairs simultaneous interactions of FGF2 with several PI(4,5)P₂ molecules and also prevents the formation of the C95 – C95 bridge. Given the critical role of C95 in FGF2 oligomerization, membrane pore formation, and secretion from cells, the simulations stress that FGF2 dimerization depends on membrane bound FGF2 monomers in the high-affinity orientation, whose stability is provided by multiple interactions with several PI(4,5)P₂ molecules. Therefore, the combined findings from these simulations predict that PI(4,5)P₂ dependent oligomerization of FGF2 is initiated through dimerization that is driven by the formation of C95 – C95 disulfide bridges. These FGF2 dimers appear to assemble into higher FGF2 oligomers driven by C77 – C77 disulfide links.

In conclusion, this study defines the minimal machinery required for FGF2 membrane translocation providing direct proof for a previously suggested model of unconventional secretion that was derived from cell-based data. This process depends on sequential and mutually exclusive interactions of FGF2 with PI(4,5)P₂ and heparan sulfates and is thermodynamically driven by FGF2 oligomerization and membrane insertion. These translocation intermediates are dynamic structures with a subunit number in the range of 8 to 12 FGF2 molecules. Therefore, depending only on two essential trans-acting factors, PI(4,5)P₂ and heparan sulfates, unconventional secretion of FGF2 is based upon a novel type of protein translocation across membranes with the cargo protein forming its own translocation intermediate by oligomerization and membrane insertion.

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[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for submitting your work entitled "Minimal Machinery Required for Unconventional Secretion of Fibroblast Growth Factor 2" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor. The reviewers have opted to remain anonymous.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

As you will see from the reviewer's comments, the biochemical reconstitution of FGF2 translocation is considered a biochemical tour de force. Discussion amongst the reviewers, however, resulted in a unanimous view that, at present, the manuscript largely confirms the molecular requirements for FGF2 translocation that were identified previously. The reviewers and I encourage you to continue this line of experimentation, and to this end, they make several suggestions that may help you to provide new information regarding the mechanism of FGF2 translocation. Specific questions that the reviewers felt were especially important to address and expand on regard the nature of the FGF2 translocation intermediate and PIP2 dynamics. Specifically, does FGF2 form a structural pore (versus simply rupturing the membrane) and, related, does PIP2 translocate with FGF2, or remain on the 'donor' leaflet? Though the reviewers concluded that suitable revisions to the manuscript would likely substantial effort, they expressed enthusiasm for considering a manuscript that does contain additional information.

Reviewer #1:

This is a superbly conducted study that investigates the translocation of FGF2 across a synthetic membrane bilayer using a GUV-based experimental system. The authors find that binding of FGF2-GFP to PIP2 is required to initiate translocation and that lumenal heparin is required to accumulate FGF2-GFP within the lumen of the vesicle. Long chain heparin is shown to effectively compete for binding to PIP2, providing an explanation for the vectoral transport (i.e., secretion) previously observed with cells. The results generally support the conclusions of the manuscript, though I think that the authors need to address a couple of points to raise the significance of the manuscript.

1) I commend the authors for a rigorous and convincing reconstitution of FGF translocation, though the manuscript essentially confirms requirements defined by published work using cell-based approaches without revealing anything fundamentally new about the mechanism of translocation. Two suggestions for enhancing the significance of the work are listed below.

2) The conclusion that FGF2-GFP forms a pore, revealed by diffusion of Alexa 647 across the GUV membrane, has important implications regarding the mechanism of translocation. The formation of a toroid intermediate was alluded to (subsection “Biochemical reconstitution of FGF2 membrane translocation with purified components”), but the results don't distinguish the formation of a bona fide pore, which in my mind is constituted by a structure of defined stoichiometry and dimensions, versus a less definite breach of membrane integrity (e.g., due to crowding on the membrane) that results in leakage of the dye across the membrane. I wonder if a systematic test of soluble tracers of varying size might reveal a size limit that would lend support to the conclusion that a pore is an intermediate structure formed during translocation.

3) The conclusion that ATP1A1 plays no core role in FGF2 translocation is not satisfying because it raises the question, does the observed translocation truly reconstitute the physiological mechanism? I realize that this is technically challenging to address, but defining the contribution of ATP1A1, be it a core role or a regulatory function, would, (a) provide new information, and/or (b) might bolster the conclusion that it is not required for translocation.

Reviewer #2:

This is an interesting report that demonstrates a possible mechanism or at least a minimal system allowing translocation of FGF2 across a lipid bilayer based on FGF2 first binding and clustering on one side of a PIP-containing membrane and then flipping over to be captured by heparin on the other side. The in vitro results demonstrating feasibility are generally clear, and the mutational analysis, especially of mutants with perturbed binding to PIP2 but not heparin are consistent with the proposed cellular mechanism. Several issues should be addressed to strength the claim that this effect is what happens in the cell.

What is missing is discussion of how FGF2 traverses the hydrophobic part of the membrane or if it always travels with PIP2 and delvers it to the other leaflet. There is extensive literature about PIP2 flipping or enhancing flipping of other lipids by e.g. Sulpice and colleagues in platelets and other systems that seems highly relevant and should be cited and discussed in this context. E.g. Bucki R, Giraud F, Sulpice (2000) Phosphatidylinositol 4,5-bisphosphate domain inducers promote phospholipid transverse redistribution in biological membranes. Biochemistry 39: 5838-5844.

Is the only binding of FGF2 to PIP2 via the headgroup? If not, the competition between IP₃ and heparin disaccharide lacks the full binding energy and it's not clear how to distinguish binding from electrostatic competition.

How specific is the effect of heparin? Is there a similar effect of hyaluronic acid, which might be more commonly bound on the outer cell surface?

How much of the interaction in vitro is due to electrostatics? How dependent is the binding affinity on ionic strength or divalent cations?

A control is needed to determine if heparin in the vesicle lumen changes the osmotic pressure inside and consequently the membrane tension.

A most interesting test would be between two vesicle types that have the same amount of PIP2 with heparin in the lumen but with different cholesterol content to shift from fully mixed to demixed bilayers, since this process is implicated in trafficking. The other controls are required technical controls but have no direct biological relevance.

Reviewer #3:

In their study, the authors reconstitute membrane translocation of FGF2 in vitro, confirming roles for PI(4,5)P₂, heparan sulfates, and FGF2 oligomerization in the process. Other factors that promote FGF2 plasma membrane translocation in vivo were not required, suggesting they function only in a regulatory manner. Overall, the study is beautifully performed, and the data presented are rigorous and quantitative. However, the major concern is that the work is largely confirmatory in nature. Please see below.

1) Sequential interactions with PI(4,5)P₂ and heparan sulfate was shown previously to be critical in FGF2 translocation across the plasma membrane. Also, the role for PI(4,5)P₂ in promoting FGF2 oligomerization and membrane pore formation was also defined previously. The work done here solidifies/confirms our understanding of FGF2 translocation, but does not really extend it. In light of this, can the authors provide any evidence as to the mechanism by which ATP1A1 regulates FGF2 translocation, even if it is not absolutely required?

2) To extend the study, could the author investigate whether the addition of appropriately positioned cysteine residues to other members of the FGF family members, which have signal peptides, enables them to be transported across membranes in vitro?

[Editors’ note: what now follows is the decision letter after the authors resubmitted for further consideration.]

Thank you for resubmitting your work entitled "Key Steps in Unconventional Secretion of Fibroblast Growth Factor 2 Reconstituted with Purified Components" for further consideration at eLife. Your revised article has been favorably evaluated by Vivek Malhotra (Senior editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors.

The manuscript has been improved and the reviewers recommend publication. You will see from the reviews below, which include the opinion of a new reviewer, that there are several points that should be addressed with additional revisions. These require only modification to the text suggested by reviewer 1 (point #3), reviewer # 2 (figure and the legend) and 3.

Reviewer #1:

The major issue raised during review of the original manuscript regarded the confirmatory nature of the manuscript, which confirmed specific requirements that were identified previously using cell based assays. In the revised manuscript, the authors now include several new findings that shed some light onto the mechanism of FGF2 translocation across a membrane. I find the most compelling of these to be the following:

1) Calibrated fluorescence measurements of labeled FGF2 was used to quantify the numbers of FGF molecules present in membrane-associated clusters. This provides some insight into possible mechanism of formation of an initial translocation intermediate.

2) Molecular dynamics simulations of FGF2 binding to PIP2, and FGF2 oligomerization, revealed possible new binding modes. Although these were not validated with 'wet' experiments, they provide some evidence to support the authors' speculation that FGF2 assembles into a larger pore-forming complex that mediates translocation.

3) The new data lead the authors to suggest a toroid translocation intermediate. I have a difficult time appreciating the manner in which FGF subunits might form a toroid intermediate, and especially how such a disulfide-bonded structure would be released from the cell, but the new data provide a foundation for this speculation. As this is not addressed in a substantial manner in the Discussion, the authors may wish to describe their thinking in a bit more detail.

Overall, my major concerns raised with the original submission have been substantially addressed.

Reviewer #2:

Fibroblast Growth Factors (FGFs) participate in a cell signaling pathways that are required for essential biological functions such as embryonic development, hematopoiesis, and wound repair. The focus of this manuscript is FGF2 which functions in normal cell growth and when mis-regulated plays a role in tumor-induced angiogenesis that is thought to be associated with tumor cell resistance to treatment therapies. FGF2 lacks a signal peptide and is transported into extracellular space by an ER/Golgi-independent mechanism. Results from cell-based experiments and in vitro studies, translocation of FGF2 is proposed to happen by direct protein translocation across the plasma membrane. This process depends on FGF2 oligomerization and sequential interaction with PI(4,5)P₂ at the inner leaflet and heparan sulfate proteoglycans at the outer surface.

In this report the authors use NMR, in vitro translocation studies, and molecular dynamic simulations to study the mechanism of FGF2 translocation across membranes. Using NMR titration and binding assays the authors show that FGF2 binding to PI(4,5)P₂ and long chain heparins are mutually exclusive and that FGF2 has higher binding affinity for heparan sulfates and long-chain heparins. Using GUVs the authors confirm that PI(4,5)P₂ and heparan sulfates are sufficient to drive some FGF2 translocation across the membrane). They also confirm that FGF2 must oligomerize for translocation. Using supported lipid bilayers and single molecule techniques FGF2, the authors find that FGF2 oligomers are heterogeneous containing a range of ~8-12 subunits. Molecular dynamic simulations of early steps of FGF2 binding to PI(4,5)P₂ confirmed that residues identified in previous mutational analysis studies are important for PI(4,5)P₂ binding and that there may be previously unknown residues involved in PI(4,5)P₂ binding when several PI(4,5)P₂ head groups bind FGF2 at the same time.

The novelty of this manuscript is the establishment of an in vitro system that has allowed the authors to directly test the minimal components FGF2 translocation. However, the results appear to confirm previous finding about FGF2 translocation that were discovered using other approaches. While solid and important work, it may not be of high enough impact for publication in eLife. The paper would be significantly strengthened if the authors were able to show that their work has led to novel, or previously unknown mechanism of FGF2 translocation.

1) It would be helpful to see the entire HSQCs for the binding titrations. Are the residues that bind PI(4,5)P₂ and Heparan the only ones that shift?

2) How did the authors confirm that the GUVs contained PI(4,5)P₂ on their surfaces and contain long-chain heparins in their lumen?

3) How does the concentration of FGF2 used in the in vitro experiments compare to the amount of FGF2 present in cells. If the amount of FGF2 added to the GUVs is lowered does translocation still occur?

4) The last part of the paper present molecular dynamics studies that suggests that there are previously uncharacterized residues on FGF2 that are important for translocation. Were these tested in the in vitro system and in cells to confirm their importance?

Reviewer #3:

The revision of this manuscript makes important changes and adds significant new data involving the roles of cholesterol, the lack of effect of heparin disaccharide in translocation, and informative new single molecule studies. As a result this already strong report is further improved and will be of interest to a range of readers. Publication in its present form would be appropriate.

There are however, three points that might be considered. Not that they need to be fully resolved before acceptance of the work, but the issues might be worth taking into account for interpretation.

First, the inability of heparin disaccharides to mimic the effect of heparin might be interpreted differently. The result is analogous to a PIP2-binding protein that does not bind IP₃ well. If the competition between PIP2 and heparin were analogous to an enzyme inhibitor and a substrate, a charge-matched heparin construct should compete for PIP2 if the concentration is enough. The need for a heparin polymer suggests that polyelectrolyte effects in addition to or instead of lock-key binding is relevant here. That was the reason to ask if HA can mimic heparin if it's packed in the vesicle lumen at high concentration. Heparin has nominally twice the charge density of HA, but maybe high concentration of HA would work. Without more data about e.g. the hydration state of the lipid and heparin bound to the FGF, it seems hard to distinguish traditional "specific" binding from equally specific charge density and polyelectrolyte effects.

Second, the experiments here are all done with only monovalent cations in the buffers. But in vivo, heparin is in a medium with mM Mg²⁺ and perhaps more important mM Ca²⁺. Most likely addition of extracellular concentration of Ca²⁺ or even intracellular Mg²⁺ makes the experiments intractable, but at least some mention of divalents and what they are known from literature to do to PIP2 and heparin would be useful.

Third, in principle some of the issues can be addressed by MD simulation, but the system here is too briefly described and possibly too limited to get into the atomic detail needed to get an accurate picture of the charge interactions. For example, when PIP2 or heparin bind FGF, are waters lost or retained at the interface of the negative oxygen of the ligand and the positive nitrogen of the protein? The fact that the net charge of PIP2 is –4 suggests that there are limits in the MD. Recent work from Radhakrishnan's group show that a fuller treatment of PIP2 under realistic conditions reduces the net charge closer to –3, which alters the way the lipid orients and binds ligands.

As mentioned above, none of these issues are probably easily resolvable and they need not delay this study. But perhaps addressing the issues would strengthen the interpretation.

[Editors’ note: the author responses to the first round of peer review follow.]

As you will see from the reviewer's comments, the biochemical reconstitution of FGF2 translocation is considered a biochemical tour de force. Discussion amongst the reviewers, however, resulted in a unanimous view that, at present, the manuscript largely confirms the molecular requirements for FGF2 translocation that were identified previously. The reviewers and I encourage you to continue this line of experimentation, and to this end, they make several suggestions that may help you to provide new information regarding the mechanism of FGF2 translocation. Specific questions that the reviewers felt were especially important to address and expand on regard the nature of the FGF2 translocation intermediate and PIP2 dynamics. Specifically, does FGF2 form a structural pore (versus simply rupturing the membrane) and, related, does PIP2 translocate with FGF2, or remain on the 'donor' leaflet? Though the reviewers concluded that suitable revisions to the manuscript would likely substantial effort, they expressed enthusiasm for considering a manuscript that does contain additional information.

We have submitted a revised version of the manuscript that contains both additional technical controls and two entirely new sections providing novel insights into the molecular mechanism of FGF2 membrane translocation. Beyond the reconstitution of FGF2 membrane translocation with purified components and the demonstration of mutually exclusive interactions of FGF2 with PI(4,5)P₂ versus heparin, we have now added a series of single molecule studies that functionally correlate the oligomeric state of PI(4,5)P₂ induced FGF2 oligomers with membrane insertion and pore formation. Furthermore, the manuscript now contains a series of atomistic molecular dynamics simulations that provide novel insights into the binding mechanism of FGF2 to PI(4,5)P₂ as well as into the molecular mechanism of the initial steps of FGF2 oligomerization triggered by PI(4,5)P₂. With these modifications, the revised manuscript addresses the three major aspects that were raised by the reviewers:

1) We have added additional controls to the core part of the original manuscript. This concerns experiments addressing questions of specificity when different forms of heparin are used as luminal traps for FGF2 in GUVs (new Figures 4 and 9) as well as a potential role of cholesterol in PI(4,5)P₂ dependent FGF2 membrane translocation (new Figures 5 and 9).

2) We have added an entirely new section providing a functional single molecule

analysis of membrane-inserted FGF2 oligomers (new Figs. 10, 11 and 12). Following the suggestions of the reviewers, these studies provide the first insights into the structure-function relationship of membrane-inserted FGF2 oligomers. Our findings demonstrate PI(4,5)P₂ dependent FGF2 oligomers to represent dynamic structures in the range of 10 to 12 subunits that form membrane-inserted translocation intermediates during unconventional secretion of FGF2. These findings are discussed in the context of a toroidal membrane pore and the assembly/disassembly hypothesis of FGF2 membrane translocation we have put forward previously [reviewed in (1)].

3) We added a new section based upon atomistic molecular dynamics simulations providing clues about the molecular mechanism of FGF2 binding to PI(4,5)P₂ and the initial steps of PI(4,5)P₂ induced oligomerization of FGF2 (new Figure 13 and 14, Figure 13—figure supplements 1-8 and Figure 14—figure supplements 1-3 and Video 1 and 2). Key aspects of this part are (i) the identification of additional binding sites in FGF2 resulting in multiple interactions of one FGF2 molecule with several PI(4,5)P₂ molecules and (ii) the identification of a high affinity dimer interface in FGF2 highlighting a disulfide bridge connecting two cysteine 95 residues. These results are discussed in the context of the FGF2 membrane translocation model we are putting forward with the current manuscript.

Reviewer #1:

1.) Oligomeric state of membrane-inserted FGF2 forms and size limit of membrane

pores generated by PI(4,5)P₂ induced FGF2 oligomers

In previous studies, we have conducted experiments analyzing a potential size cutoff of membrane pores generated by PI(4,5)P₂ dependent FGF2 oligomerization and membrane insertion (2). While we found an Alexa488 dye (1 kDa) to physically traverse the membrane, the same dye coupled to either a dextran molecule (10 kDa) or cytochrome c (12 kDa) did not. Furthermore, we have previously shown that, upon membrane insertion of FGF2 oligomers, transbilayer diffusion of membrane lipids occurs providing direct evidence for the formation of a toroidal pore (2). These findings suggest that FGF2 oligomers insert into membranes through electrostatic interactions with PI(4,5)P₂ thereby opening up a lipidic pore with a toroidal structure. In the revised manuscript, we have now included a series of single molecule measurements using both GUVs and supported lipid bilayers functionally correlating the oligomeric state of FGF2 with membrane pore formation (new Figures 10, 11 and 12). We find membrane-inserted FGF2 oligomers to represent dynamic structures with pore forming species characterized by about 10 to 12 FGF2 subunits. As discussed in the revised manuscript, these findings are consistent with a toroidal architecture and point to a dynamic structure that is continuously assembled at the inner leaflet and disassembled at the outer leaflet resulting in net transport of FGF2 molecules across the membrane.

2) The role of ATP1A1 in FGF2 membrane translocation

The fact that FGF2 membrane translocation can be observed in the absence of ATP1A1 does not imply that our reconstitution system is unrelated to the physiological mechanism. The most simple explanation would be that ATP1A1 is required in cells as a first physical contact to accumulate FGF2 at the inner leaflet of the plasma membrane at sites that provide proximity to Tec kinase and/or PI(4,5)P₂. This requirement might be simply bypassed in the reconstitution system when the essential components are present in a purified form. However, provided ATP1A1 could be included in a proper way into our reconstitution system, it is possible that the efficiency of FGF2 membrane translocation would increase. Therefore, while ATP1A1 clearly is not essential for FGF2 membrane translocation to occur, we are indeed investigating ways of reconstituting ATP1A1 as part of our FGF2 membrane translocation assay. However, as noted by reviewer #1, reconstituting multi-subunit integral membrane proteins in a functional form is technically challenging and beyond the focus of the current manuscript.

Reviewer #2:

[…] 1) How does FGF2 traverse the hydrophobic part of the membrane?

The results from various studies point at a toroidal architecture of FGF2 induced membrane pores where the FGF2 oligomer is located in the center with each monomer bound to PI(4,5)P₂ [reviewed in (1)]. The combined findings led to the assembly/disassembly model of FGF2 membrane translocation where FGF2 subunits are added to membrane-inserted FGF2 oligomers at the inner leaflet followed by removal of FGF2 subunits at the outer leaflet. The latter step is mediated by heparan sulfates that outcompete PI(4,5)P₂ with regard to binding to FGF2. This process results in directional transport across the plasma membrane along with FGF2 trapping in the extracellular space where FGF2 remains bound to heparan sulfates on cell surfaces. As also explained in our response to reviewer #1, this view is supported by several lines of evidence. First, a toroidal architecture of FGF2 membrane pores is indicated by transbilayer diffusion of membrane lipids observed following membrane insertion of FGF2 oligomers (2). These results were corroborated by findings demonstrating that FGF2 oligomers can be extracted from membranes using carbonate, a direct indication of electrostatic rather than hydrophobic interactions with the membrane (3). Finally, the assembly/disassembly model as part of a toroidal pore is strongly supported by the current study demonstrating mutually exclusive interactions of FGF2 with PI(4,5)P₂ and heparin, the latter mimicking cellular heparan sulfates outcompeting PI(4,5)P₂ and thereby trapping FGF2 at the extracellular leaflet. In conclusion, all data from various studies point to a mechanism of FGF2 membrane translocation that is characterized by a lack of physical contacts of FGF2 with the hydrophobic core of the membrane. With regard to transbilayer diffusion of membrane lipids (2), it is indeed likely that this phenomenon includes PI(4,5)P₂ itself as part of the assembly/disassembly mechanism of FGF2 membrane translocation. As suggested by reviewer #2, in the revised manuscript these aspects are now discussed in more detail along with the relevant literature on domain induced PIP2 flipping in cellular membranes.

2) Does FGF2 exclusively bind to the headgroup of PI(4,5)P2 ?

There is no evidence that FGF2 interacts with the hydrophobic parts of PI(4,5)P₂. Using atomistic molecular dynamics simulations, we have further tested this possibility and, consistent with previous biochemical and structural data, found a high affinity binding site for the headgroup of PI(4,5)P₂. (new Figure 13 and 14, Figure 13—figure supplements 1-8 and Figure 14—figure supplements 1-3 and Video 1 and 2). Intriguingly, these studies revealed additional binding sites in FGF2 for PI(4,5)P₂, suggesting that FGF2 accumulates PI(4,5)P₂ molecules at the sites of oligomerization and membrane pore formation. These findings are of extraordinary relevance for the formation of membrane pores with a toroidal architecture as we have shown previously that PI(4,5)P₂ is required for FGF2 membrane insertion based upon its ability to stabilize the high membrane curvature that characterizes toroidal pores (2). The molecular dynamics studies included into the revised manuscript further provide clues on the molecular mechanism of the initial steps of PI(4,5)P₂ induced FGF2 oligomerization which will be highly relevant for future studies analyzing the fine structure of FGF2 oligomers as part of toroidal pores.

3) How specific is the effect of heparin? Could it be replaced by for example hyarulonic acid?

FGF family members are well characterized with regard to their ligand binding specificities. FGF2 has been shown to interact exclusively with sulphated glycosaminoglycans as contained in heparan sulfate proteoglycans. As opposed to heparan sulfates and other sulfated proteoglycans, cellular forms of hyaluronic acid do not contain sulfate groups. In any case, reviewer #2 inspired us to systematically compare long-chain heparins with a defined heparin disaccharide (as used in Figures 1 and 2) in FGF2 membrane translocation assays. As shown in the new Figure 4 and quantified in the new Figure 9, replacing long-chain heparins by a heparin disaccharide does not have any impact on membrane pore formation, however, FGF2 membrane translocation is almost fully blocked. These findings are in line with the NMR data and biochemical experiments shown in Figures 1 and 2 demonstrating long-chain heparins to be required to outcompete PI(4,5)P₂ with regard to binding to FGF2. Our combined results therefore corroborate the proposed model for the molecular mechanism of directional membrane translocation of FGF2 based on sequential interactions with PI(4,5)P₂ and heparan sufates on opposing sides of the membrane.

4) Does heparin change the osmotic pressure and, in turn, membrane tension?

As explained in “Materials and methods”, the generation procedures of all types and batches of GUVs were controlled by osmolality measurements and demonstrated not to be affected by adding either long-chain or disaccharide heparins. Therefore, artifacts related to differences in osmotic pressure and membrane tension can be excluded.

5) Does cholesterol play a role in FGF2 membrane translocation?

As suggested by reviewer #2, we have conducted reconstitution experiments with heparin-containing GUVs that either contained or lacked cholesterol. As shown in the new Figure 5 and quantified in the new Figure 9, omission of cholesterol causes a substantial decrease of both membrane pore formation and translocation of FGF2. The experiments shown in Figure 5 indicate that, in the absence of cholesterol, binding of FGF2 to PI(4,5)P₂ is strongly impaired. This is consistent with previous studies from our laboratory demonstrating strong binding of FGF2 to PI(4,5)P₂ in liposomes with a plasma membrane like lipid composition whereas binding was found poor when PI(4,5)P₂ was reconstituted in pure PC liposomes (4,5). Our combined findings suggest that components of the FGF2 secretion apparatus such as PI(4,5)P₂ are organized in cholesterol dependent microdomains that are required for efficient recruitment, oligomerization and membrane translocation of FGF2. The new results are shown in Figures 5 and 9 in the revised manuscript and are discussed accordingly.

Reviewer #3:

1) What could be the role of ATP1A1 as a novel aspect of FGF2 membrane translocation?

In previous studies, we have demonstrated a direct interaction of FGF2 with the cytoplasmic domain of ATP1A1 (6). As explained in our response to reviewer #1, the function of ATP1A1 in FGF2 secretion from cells might be a first physical contact of FGF2 with the inner leaflet of plasma membranes. One possible explanation for a requirement of ATP1A1 in FGF2 secretion from cells might be a need to accumulate FGF2 in plasma membrane microdomains mediating proximity to additional components such as Tec kinase and PI(4,5)P₂. In our reconstitution system, this function might be bypassed when the essential components are present in a purified form. While we do run projects analyzing the function of ATP1A1 both in a cellular context and in reconstitution systems, these studies are clearly beyond the focus of the current manuscript.

2) Do cysteine residues transplanted to FGF family members with signal peptides promote membrane translocation similar to FGF₂?

We have done such experiments in the context of cells (7). As a model FGF protein carrying a signal peptide, FGF4 has been used as it is a close structural relative of FGF2. Following removal of the signal peptide of FGF4, secretion became undetectable, however, after transplanting the critical cis-elements including Cys77 and Cys95, the corresponding FGF4/2 hybrid protein was indeed found rerouted into the unconventional secretory pathway of FGF2 (7). Accordingly, a double cysteine mutant of FGF2 (C77/95A) was used in the current study and demonstrated to be strongly impaired in membrane translocation (Figures 7 and 9).

References:

1. La Venuta, G., Zeitler, M., Steringer, J. P., Müller, H. M., and Nickel, W. (2015) The Startling Properties of Fibroblast Growth Factor 2: How to Exit Mammalian Cells without a Signal Peptide at Hand. J Biol Chem 290, 27015-27020.

2. Steringer, J. P., Bleicken, S., Andreas, H., Zacherl, S., Laussmann, M., Temmerman, K., Contreras, F. X., Bharat, T. A., Lechner, J., Müller, H. M., Briggs, J. A., Garcia-Saez, A. J., and Nickel, W. (2012) Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P₂)-dependent Oligomerization of Fibroblast Growth Factor 2 (FGF2) Triggers the Formation of a Lipidic Membrane Pore Implicated in Unconventional Secretion. J Biol Chem 287, 27659-27669.

3. Zeitler, M., Steringer, J. P., Müller, H. M., Mayer, M. P., and Nickel, W. (2015) HIV-Tat Protein Forms Phosphoinositide-dependent Membrane Pores Implicated in Unconventional Protein Secretion. J Biol Chem 290, 21976-21984.

4. Temmerman, K., Ebert, A. D., Müller, H. M., Sinning, I., Tews, I., and Nickel, W. (2008) A direct role for phosphatidylinositol-4,5-bisphosphate in unconventional secretion of fibroblast growth factor 2. Traffic 9, 1204-1217.

5. Temmerman, K., and Nickel, W. (2009) A novel flow cytometric assay to quantify interactions between proteins and membrane lipids. J Lipid Res.

6. Zacherl, S., La Venuta, G., Müller, H. M., Wegehingel, S., Dimou, E., Sehr, P., Lewis, J. D., Erfle, H., Pepperkok, R., and Nickel, W. (2015) A direct role for ATP1A1 in unconventional secretion of fibroblast growth factor 2. J Biol Chem 290, 3654-3665.

7. Müller, H. M., Steringer, J. P., Wegehingel, S., Bleicken, S., Munster, M., Dimou, E., Unger, S., Weidmann, G., Andreas, H., Garcia-Saez, A. J., Wild, K., Sinning, I., and Nickel, W. (2015) Formation of Disulfide Bridges Drives Oligomerization, Membrane Pore Formation and Translocation of Fibroblast Growth Factor 2 to Cell Surfaces. J Biol Chem 290, 8925-8937.

[Editors' note: the author responses to the re-review follow.]

[…] Reviewer #1:

The major issue raised during review of the original manuscript regarded the confirmatory nature of the manuscript, which confirmed specific requirements that were identified previously using cell based assays. In the revised manuscript, the authors now include several new findings that shed some light onto the mechanism of FGF2 translocation across a membrane. I find the most compelling of these to be the following:

1) Toroidal translocation intermediate

A lipidic membrane pore with a toroidal architecture containing the FGF2 oligomer in its center is supported by several independent lines of evidence from previous publications and data from our current work. However, the precise molecular mechanism by which membrane-inserted forms of FGF2 oligomers act as translocation intermediates is not fully understood. We have put forward the hypothesis that membrane-inserted FGF2 oligomers are dynamic structures to which FGF2 subunits are constantly added at the inner leaflet concomitant with the removal of subunits at the outer leaflet. In this way, at steady state, membrane-inserted forms of FGF2 have a dynamic oligomeric state in the range of 8 to 12 FGF2 subunits that are continuously remodeled by adding and removing subunits on opposing sides of the membrane. It will be a challenge for future studies to find out whether these assembly/disassembly units are monomers or disulfide-bridged dimers of FGF2, the latter being functional in FGF2 signaling as part of ternary complexes with heparan sulfates and FGF high affinity receptors. Furthermore, a big goal in our lab will be to elucidate the three-dimensional architecture of membrane inserted FGF2 oligomers to fully understand how these complexes are functioning as translocation intermediates in unconventional secretion of FGF2.

As requested by reviewer 1, we have modified the discussion to explain these aspects in more detail.

2) Modifications to Fig. 10 and the corresponding legend

We have streamlined Figure 10 with a simplified labelling of its subpanels that makes this figure more self-explanatory. Furthermore, the lipid tracer is now shown for both variant forms of FGF2 which helps to better distinguish the two experimental conditions being compared. The legend has been adapted accordingly.

Reviewer #2:

1) HSQC data from NMR experiments

The full set of HSQC data from the experiments shown in Figure 1 has been added to the source file with all raw data available to the reader.

2) Measurements of PI(4,5)P2 and heparin incorporation into GUVs

Luminal incorporation of heparin into GUVs was monitored by confocal microscopy in control experiments using a fluorescent derivative. Likewise, the presence of PI(4,5)P₂ in the bilayers of GUVs was analyzed using a recombinant fusion protein consisting of GFP and the PH domain of PLCδ1, a canonical PI(4,5)P₂ marker. The ‘Materials and methods’ section has been modified accordingly along with the corresponding references.

3) How does the concentration of FGF2 in cells compare with FGF2 concentrations usedin reconstitution experiments?

Though the precise intracellular concentrations of FGF2 in μM has not been reported in the literature, tumor cells massively overexpress FGF2 under pathophysiological conditions which drives an autocrine secretion/signaling loop of FGF2. In our reconstitution experiments, FGF2 is used at low concentrations between 50 and 200 nM (to allow for single molecule measurements) that are likely to be way below the intracellular concentration of FGF2 in tumor cells.

4) Analysis of additional residues in FGF2 with affinity to PI(4,5)P2 as identified by MD simulations

The residues we have identified previously in biochemical experiments (K127, R128 and K133) form a high affinity binding pocket for PI(4,5)P₂ [Temmerman et al., (2008) Traffic 9:1204-1217]. Consistent with both biochemical experiments and MD simulations, these residues are essential for interactions of FGF2 with PI(4,5)P₂ containing membranes and are required for FGF2 secretion from cells. The identification of additional residues in FGF2 mediating PI(4,5)P₂ clustering as identified in MD simulations is a novel finding of the current work with important implications for future studies. However, their detailed analysis in reconstitution experiments and cell-based assays is beyond the scope of the current manuscript.

Reviewer #3:

1) Electrostatic aspects of mutually exclusive interactions of FGF2 with various forms of heparin and PI(4,5)P₂

The electrostatic determinants of FGF2 binding to PI(4,5)P₂, heparin isaccharides and long-chain heparins are an important aspect with regard to the mechanism of FGF2 membrane translocation we are proposing. However, it is clear from the literature that all of these interactions are not merely based upon the number of electrostatic pairs. Rather, both the orientation of sugar subunits in the heparin backbone and the positioning of sulfate moieties along the ring structure are critical determinants for FGF2 binding. There are in fact examples for different kinds of heparin molecules that are identical in net charge but differ in the positioning of the sulfate groups resulting in strong binding versus a complete lack of FGF2 binding [Li et al., (2014) ACS Chem Biol 9:1712-1717) and papers therein]. Likewise, we have previously demonstrated that FGF2 binding to PI(4,5)P₂ is much stronger compared to PI(3,4,5)P3 [Temmerman et al., (2008) Traffic 9:1204-1217], pointing at a defined binding pocket for PI(4,5)P₂ in FGF2 as demonstrated by our NMR experiments. Therefore, hyaluronic acid that lacks sulfate groups even at high concentrations is very unlikely to outcompete PI(4,5)P₂ with regard to binding to FGF2. As outlined in our manuscript, there are two defined and overlapping binding epitopes in FGF2 for heparin and PI(4,5)P₂ resulting in mutually exclusive interactions, a key aspect of the translocation model we are proposing.

2) Potential effects of divalent cations on FGF2-heparin interactions

As indicated by reviewer 3, the use of divalent calcium and magnesium ions in biochemical reconstitution experiments employing GUVs is technically challenging. However, in cell-based FGF2 secretion experiments, we found that both calcium (1.8mM) and magnesium ions (0.8 mM) in the extracellular medium does not affect the ability of heparan sulfates to trigger FGF2 membrane translocation and to retain FGF2 on cell surfaces (Zehe et al., 2006, Proc. Natl. Acad. Sci. U.S.A. 103:15479-15484). These observations in intact cells demonstrate that the mechanism we are proposing is functional even in the presence of millimolar concentrations of divalent cations.

3) MD simulations of the interaction between FGF2 and PI(4,5)P₂.

Reviewer 3 commented on the net charge of PI(4,5)P₂ and referred to recent work by the Radhakrishnan group [e.g., J Phys Chem B 117, 8322 (2013)]. Their quantum mechanical calculations indicated a net charge of PI(4,5)P₂ to be –4 around pH 7. This is fully consistent with our work. However, deviations from the net charge of –4 were suggested in the presence of divalent ions. While the effects of divalent salt and its influence on the net charge of PI(4,5)P₂ could be explored in MD simulations, it would require a computational resource that is larger by a factor of 5–10 compared to the resources used for the present work. In addition, the nature of the results is not expected to change as both –3 and –4 are exceptionally large charges for a lipid head group and will be predominate over any other lipid in terms of electrostatic interactions.

Another aspect raised by reviewer 3 concerned the role of water molecules in the course of FGF2 binding to PI(4,5)P₂.To address this question, we analyzed our simulation data carefully by computing radial distribution functions, solvent accessible surface areas, number of water molecules around the PI(4,5)P₂ binding pocket in FGF2 and number of hydrogen bonds between FGF2 binding pocket residues and water molecules. The best overall picture is given by the number of water molecules around the binding pocket region (residues K127, R128, K133). As shown in Author response image 1, the number of water molecules is about 95 when FGF2 is not in contact with PI(4,5)P₂:

Author response image 1

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When PI(4,5)P₂binds to FGF2 by occupying the binding pocket at about 500 ns, the number of water molecules around the binding pocket region decreases by ~25% to about 70. This is quite expected, since the PI(4,5)P₂ head group blocks some of the water molecules away from the binding pocket region. Nonetheless, the binding pocket remains very hydrated, i.e. the hydration level does not change in any essential manner.

Author details

1. Julia P Steringer

   Heidelberg University Biochemistry Center, Heidelberg, Germany

   Contribution

   JPS, Conceptualization, Data curation, Investigation

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-9418-2762

2. Sascha Lange

   Institut für Chemie und Biochemie, Freie Universität Berlin, Berlin, Germany

   Present address

   Department of Molecular Biophysics, Leibniz-Forschungsinstitut fürMolekulare Pharmakologie, Berlin, Germany

   Contribution

   SL, Conceptualization, Data curation, Investigation, Methodology

   Contributed equally with

   Sabína Čujová, Radek Šachl, Chetan Poojari, Fabio Lolicato, Oliver Beutel, Ünal Coskun, Alf Honigmann, Ilpo Vattulainen, Martin Hof and Christian Freund

   Competing interests

   The authors declare that no competing interests exist.

3. Sabína Čujová

   J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic

   Contribution

   SČ, Data curation, Investigation, Methodology

   Contributed equally with

   Sascha Lange, Radek Šachl, Chetan Poojari, Fabio Lolicato, Oliver Beutel, Ünal Coskun, Alf Honigmann, Ilpo Vattulainen, Martin Hof and Christian Freund

   Competing interests

   The authors declare that no competing interests exist.

4. Radek Šachl

   J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic

   Contribution

   RŠ, Conceptualization, Data curation, Formal analysis, Investigation, Methodology

   Contributed equally with

   Sascha Lange, Sabína Čujová, Chetan Poojari, Fabio Lolicato, Oliver Beutel, Ünal Coskun, Alf Honigmann, Ilpo Vattulainen, Martin Hof and Christian Freund

   Competing interests

   The authors declare that no competing interests exist.

5. Chetan Poojari

   1. Department of Physics, University of Helsinki, Helsinki, Finland
   2. Department of Physics, Tampere University of Technology, Tampere, Finland

   Contribution

   CP, Data curation, Investigation, Methodology

   Contributed equally with

   Sascha Lange, Sabína Čujová, Radek Šachl, Fabio Lolicato, Oliver Beutel, Ünal Coskun, Alf Honigmann, Ilpo Vattulainen, Martin Hof and Christian Freund

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-6575-221X

6. Fabio Lolicato

   1. Department of Physics, University of Helsinki, Helsinki, Finland
   2. Department of Physics, Tampere University of Technology, Tampere, Finland

   Contribution

   FL, Data curation, Formal analysis, Investigation, Methodology

   Contributed equally with

   Sascha Lange, Sabína Čujová, Radek Šachl, Chetan Poojari, Oliver Beutel, Ünal Coskun, Alf Honigmann, Ilpo Vattulainen, Martin Hof and Christian Freund

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-7537-0549

7. Oliver Beutel

   Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

   Contribution

   OB, Data curation, Methodology

   Contributed equally with

   Sascha Lange, Sabína Čujová, Radek Šachl, Chetan Poojari, Fabio Lolicato, Ünal Coskun, Alf Honigmann, Ilpo Vattulainen, Martin Hof and Christian Freund

   Competing interests

   The authors declare that no competing interests exist.

8. Hans-Michael Müller

   Heidelberg University Biochemistry Center, Heidelberg, Germany

   Contribution

   H-MM, Data curation, Methodology, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

9. Sebastian Unger

   Heidelberg University Biochemistry Center, Heidelberg, Germany

   Contribution

   SU, Data curation, Methodology

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-2525-8502

10. Ünal Coskun

    Paul Langerhans Institute Dresden, Dresden, Germany

    Contribution

    ÜC, Conceptualization, Resources, Writing—review and editing

    Contributed equally with

    Sascha Lange, Sabína Čujová, Radek Šachl, Chetan Poojari, Fabio Lolicato, Oliver Beutel, Alf Honigmann, Ilpo Vattulainen, Martin Hof and Christian Freund

    Competing interests

    The authors declare that no competing interests exist.

    "This ORCID iD identifies the author of this article:" 0000-0003-4375-3144

11. Alf Honigmann

    Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

    Contribution

    AH, Conceptualization, Data curation, Methodology, Writing—review and editing

    Contributed equally with

    Sascha Lange, Sabína Čujová, Radek Šachl, Chetan Poojari, Fabio Lolicato, Oliver Beutel, Ünal Coskun, Ilpo Vattulainen, Martin Hof and Christian Freund

    Competing interests

    The authors declare that no competing interests exist.

12. Ilpo Vattulainen

    1. Department of Physics, University of Helsinki, Helsinki, Finland
    2. Department of Physics, Tampere University of Technology, Tampere, Finland
    3. MEMPHYS – Center for Biomembrane Physics, University of Southern Denmark, Denmark, United Kingdom

    Contribution

    IV, Conceptualization, Resources, Data curation, Methodology, Writing—review and editing

    Contributed equally with

    Sascha Lange, Sabína Čujová, Radek Šachl, Chetan Poojari, Fabio Lolicato, Oliver Beutel, Ünal Coskun, Alf Honigmann, Martin Hof and Christian Freund

    Competing interests

    The authors declare that no competing interests exist.

    "This ORCID iD identifies the author of this article:" 0000-0001-7408-3214

13. Martin Hof

    J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic

    Contribution

    MH, Conceptualization, Resources, Methodology, Writing—review and editing

    Contributed equally with

    Sascha Lange, Sabína Čujová, Radek Šachl, Chetan Poojari, Fabio Lolicato, Oliver Beutel, Ünal Coskun, Alf Honigmann, Ilpo Vattulainen and Christian Freund

    Competing interests

    The authors declare that no competing interests exist.

14. Christian Freund

    Institut für Chemie und Biochemie, Freie Universität Berlin, Berlin, Germany

    Contribution

    CF, Conceptualization, Data curation, Writing—review and editing

    Contributed equally with

    Sascha Lange, Sabína Čujová, Radek Šachl, Chetan Poojari, Fabio Lolicato, Oliver Beutel, Ünal Coskun, Alf Honigmann, Ilpo Vattulainen and Martin Hof

    Competing interests

    The authors declare that no competing interests exist.

15. Walter Nickel

    Heidelberg University Biochemistry Center, Heidelberg, Germany

    Contribution

    WN, Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing

    For correspondence

    walter.nickel@bzh.uni-heidelberg.de

    Competing interests

    The authors declare that no competing interests exist.

    "This ORCID iD identifies the author of this article:" 0000-0002-6496-8286

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