Lipid Binding Controls Dimerization of the Coat Protein p24 Transmembrane Helix

Please cite this article in press as: Pannwitt et al., Lipid Binding Controls Dimerization of the Coat Protein p24 Transmembrane Helix, Biophysical Journal (2019), https://doi.org/10.1016/j.bpj.2019.09.021

Article

Lipid Binding Controls Dimerization of the Coat Protein p24 Transmembrane Helix Stefanie Pannwitt,1 Michael Stangl,1 and Dirk Schneider1,* 1

Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Mainz, Germany

ABSTRACT Coat protein (COP) I and COP II complexes are involved in the transport of proteins between the endoplasmic reticulum and the Golgi apparatus in eukaryotic cells. The formation of COP I/II complexes at membrane surfaces is an early step in vesicle formation and is mastered by p24, a type I transmembrane protein. Oligomerization of p24 monomers was suggested to be mediated and/or stabilized via interactions within the transmembrane domain, and the p24 transmembrane helix appears to selectively bind a single sphingomyelin C18:0 molecule. Furthermore, a potential cholesterol-binding sequence has also been predicted in the p24 transmembrane domain. Thus, sphingomyelin and/or cholesterol binding to the transmembrane domain might directly control the oligomeric state of p24 and, thus, COP vesicle formation. In this study, we show that sequence-specific dimerization of the p24 transmembrane helix is mediated by a LQ7 motif, with Gln187 being of special importance. Whereas cholesterol has no direct impact on p24 dimerization, binding of the sphingolipid can clearly control dimerization of p24 in rigid membrane regions. We suggest that specific binding of a sphingolipid to the p24 transmembrane helix affects p24 dimerization in membranes with increased cholesterol contents. A clearly defined p24 dimerization propensity likely is crucial for the p24 activity, which involves shuttling in between the endoplasmic reticulum and the Golgi membrane, in which cholesterol and SM C18:0 concentrations differ.

SIGNIFICANCE Biological membranes contain many different lipids and proteins. Interaction of membrane proteins often is crucial for the protein’s activity, and the interaction propensity might be controlled by the lipid environment. Here, we analyzed how the binding of individual lipids and helix dimerization cooperate. We tested dimerization of the eukaryotic p24 protein, which is involved in material transfer between different intracellular membrane structures. Analyses in model membrane systems demonstrate that the binding of a defined lipid species to the protein’s transmembrane region affects interaction of the p24 protein. Specific binding of a defined lipid molecule to a single transmembrane a-helix can thus control the formation of higher-ordered protein structures and, finally, entire cellular events.

INTRODUCTION Membrane vesicles are involved in intracellular material transport between cell membranes. Vesicle formation involves defined coat protein complexes, and the thus far best characterized complexes are clathrin, coat protein I (COP I), and COP II (1). COP I and COP II complexes are involved in the transport of cargo proteins between the endoplasmic reticulum (ER) and the Golgi apparatus in eukaryotic cells, and whereas COP I vesicles mediate the retrograde vesicle transport from the Golgi to the ER and the intra Golgi transport, COP II vesicles facilitate the anterSubmitted February 21, 2019, and accepted for publication September 9, 2019. *Correspondence: [email protected] Editor: Kalina Hristova. https://doi.org/10.1016/j.bpj.2019.09.021

ograde transport from the ER to the Golgi (2). Formation of COP I/II complexes at membrane surfaces is an early step in COP I/II vesicle formation and is mastered by several proteins including p24, a type I transmembrane (TM) protein with a molecular mass of
24 kDa (2). Several p24 proteins form a protein family in eukaryotes that can further be divided into four subfamilies (a, b, g, d) (2). The p24 protein we analyze and discuss here is the protein p24b1 (also named TMED2, or p24 or p24a) and will simply be called p24 in the following. p24 proteins have a large globular N-terminal GOLD (Golgi dynamics) domain located at the luminal side of the membrane, which is followed by a coiled-coil region, a single TM a-helix, and a short C-terminal cytoplasmic tail, which is involved in the binding of other COPs as well as the small GTPase Arf1 (2). p24 can selectively bind a single sphingomyelin (SM) C18:0 molecule within the TM region, and both

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the headgroup as well as the hydrophobic acyl chain regions of the lipids appear to determine the binding specificity (3). The 181VxxTLxxIY189 amino acid motif in the p24 TM helix has been identified to be involved in SM binding, and based on molecular modeling, the SM C18:0 acyl chains fit perfectly into a cavity formed by the residues Val181, Thr184, and Leu185. In fact, the mutation of a single residue in this cavity to an amino acid with a bulkier side chain completely abolished SM C18:0 binding to the p24 TM helix (3). Besides the SM-binding motif, a potential cholesterolbinding sequence (187QIY189) has also been identified in the p24 TM domain via computational analyses (4). Noteworthy, this potential cholesterol-binding region does partially overlap with the SM C18:0 signature sequence. Depending on their subcellular localization, p24 proteins form homo- or heterodimers with other proteins of the p24 family, a crucial step in COP vesicle formation (5). Although protein dimerization was suggested to involve the coiled-coil and the cytoplasmic regions of p24 (6–8), more recent results suggest that the interaction of p24 monomers is mediated and/or stabilized by the TM helices via a rather polar, yet undefined, dimerization interface (3). Although the dimerization interface in the p24 TM helix has not been identified yet, in this study, we have identified a LQ7 motif that controls dimerization of the p24 TM helix. However, p24 TM helixhelix interactions appear to be linked to sphingolipid binding, and it has been suggested that SM binding controls p24 dimerization (3,9), which potentially results in the recruitment of the COP complex at the membrane surface in vivo (3). Thus, the p24 TM helices have an intrinsic propensity to dimerize, and the isolated helices potentially bind sphingolipids or cholesterol or both. However, here, we show that cholesterol does not directly affect p24 TM helix dimerization, albeit cholesterol-induced ordering of the lipid bilayer indirectly stabilizes p24 TM helix dimers. Binding of SM to the p24 TM helix does not affect helix dimerization in fluid but destabilizes p24 helix dimers in more ordered membrane systems. As the SM and cholesterol concentrations differ between the ER and the Golgi (10,11), the SM and cholesterol concentrations within a given membrane system might directly influence the secretory pathway by controlling the oligomeric state of p24 (i.e., COP complex formation). Because the ratio between monomeric and dimeric p24 is similar in the ER and the Golgi membrane (5), SM binding to the p24 TM helix likely counteracts dimerization of p24 caused by the increased cholesterol concentration in the Golgi, and it thereby controls the p24 monomer/dimer ratio. MATERIALS AND METHODS TOXCAT measurement Sequence-dependent dimerization of the p24 TM helix was analyzed using the TOXCAT assay (12). The p24 TM region (SRVVLWSFFEALVLVAMTLGQIYYL) was genetically fused to the DNA-binding domain of the Vibrio cholera ToxR domain, a dimeriza-

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tion-dependent transcriptional activator, and at the TMs C-terminus, the maltose-binding protein MalE of Escherichia coli was fused. Association of the expressed chimeric proteins results in ToxR-mediated activation of a reporter gene encoding chloramphenicol acetyltransferase (CAT) under the control of the ctx promoter. Construction of the TOXCAT plasmid pccKAN and the derivatives encoding the TM domain of human glycophorin A (GpA) wild-type (wt) (pccGpA-wt) and GpA G83I (pccGpA-G83I) was described previously (12). Additional pccbased plasmids were produced by ligating chemically synthesized oligonucleotide cassettes, which encode for the respective TM helix, into the NheI and BamHI restriction-digested plasmid pccKAN (12). Correct insertion of the genes was verified by DNA sequencing. Plasmids were transformed into malE-deficient E. coli strain NT326 (13), and CAT activities were determined as described (14,15). Interaction propensities were determined by measuring the CAT activity three times for at least five independent clones. To estimate the abundance of the expressed fusion proteins via Western blot analyses, whole cell lysates were probed using a polyclonal antiserum that recognized the maltose-binding protein (New England BioLabs, Frankfurt, Germany). Furthermore, for each expressed chimeric protein, proper membrane insertion and orientation was tested. The plasmids were transformed into malE-deficient E. coli NT326 cells, and growth on M9 medium with 0.2% maltose or glucose as the only hydrocarbon source was followed. The malE deficiency results in an inability of the NT326 strain to transport maltose into the cytoplasm. If the expressed chimeric proteins are inserted into the E. coli cytoplasmic membrane with the MalE domain facing the periplasm, the MalE domain will compensate the deficiency, E. coli NT326 cells will be able to grow on maltose-containing M9 minimal medium, and the topology is established.

Peptide synthesizing and labeling Peptides were custom synthesized, labeled, and purified (Peptide Specialty Laboratories, Heidelberg, Germany). Peptides corresponding to the human p24 wt (166NSRVVLWSFFEALVLVAMTLGQIYYLKRFFEVRR199), p24 Q187A (166NSRVVLWSFFEALVLVAMTLGAIYYLKRFFEVRR199), and p24 L185F (166NSRVVLWSFFEALVLVAMTFGQIYYLKRFFEVRR199) TM sequence were labeled with either ATTO 488 (Fo¨rster resonance energy transfer [FRET] donor) or ATTO 590 (FRET acceptor) at the N-terminus. The human GpA TM helix (69SEPEITLIIFGVMAGVIGTILLISYGIRRLIKK101) was labeled at the N-terminus with fluorescein (FL) or 5-6-carboxythodamine (TAMRA), respectively (16). Purity higher than 95% of the labeled peptides was confirmed by high-performance liquid chromatography and mass spectrometry. Peptides were dissolved in 2,2,2-trifluoroethanol obtained from Sigma-Aldrich (Munich, Germany). Concentrations of peptide stock solutions were determined from absorbance measurements using a PerkinElmer Lambda 35 ultraviolet/visible spectrophotometer (Waltham, MA).

FRET measurements For FRET measurements, 0.5 mM lipid (Avanti Polar Lipids, Alabaster, AL) were dissolved in chloroform/methanol (2:1) and labeled peptides in trifluorethanol. The molar ratios of labeled p24 TM helices in E. coli polar lipid extract (EPL) was 1:2000 (i.e., 0.5 mM lipid and 0.25 mM peptide) in 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC)derived liposomes 1:5000 (i.e., 0.5 mM lipid and 0.1 mM peptide). Labeled GpA TM helices were used in a molar ratio of 1:4000. Dissolved peptides and lipids were mixed, and the organic solvents were removed by a gentle stream of nitrogen. Residual solvent traces were removed by vacuum desiccation for at least 2 h. The dried peptide/lipid film was hydrated with buffer (10 mM Hepes (pH 7.4) and 150 mM NaCl) followed by five freeze-thaw cycles. Steady-state fluorescence measurements were performed at 25 C using a Horiba Fluoromax 4 system. The excitation wavelength for FL-GpA was set to 439 nm, and emission spectra were

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Lipid Binding Controls p24 Dimerization recorded from 480 to 650 nm (16) with the band-pass filters set to 5 nm. ATTO 488 was excited at 467 nm, and fluorescence emission from Atto 580-labeled p24 TM helices was also recorded from 480 to 650 nm with the band-pass filters set to 3 nm. To record excitation spectra of ATTO 488 and ATTO 580, the emission wavelength was set to 570 or 660 nm, respectively. Excitation spectra were then recorded from 300– 560 to 350–650 nm, respectively, with band-pass filters set to 3 nm. For steady-state fluorescence measurements in SOPC-derived unilamellar liposomes, the average of three mean values of three independent measurements are given. For EPL liposomes and donor acceptor titrations, mean values of three independent replicates are given. Temperature gradients were recorded every 2 C from 5 to 25 C with 1 min equilibration time and temperature tolerance of 1 C. Here, mean values of three independent experiments are given. Energy transfer E was calculated by using the donor/acceptor fluorescence intensity ratio. The lipid/peptide ratio was kept constant; each sample contained equal amounts of donor- and acceptor-labeled TM helices. Ratiometric FRET was calculated by E ¼ IA/(ID þ IA), where ID and IA are the fluorescence intensity of donor and acceptor maxima, respectively. Because the donor and acceptor emission spectra of TAMRA and FL overlap significantly, the emission spectra of labeled GpA TM helices had to be corrected for proper evaluation. To obtain solely acceptor emission, a donor spectrum had to be fitted into the donor/acceptor emission spectra and had to be subtracted.

Laurdan fluorescence emission measurements To determine lipid packing in various unilamellar liposomes, 0.2 mol % Laurdan, dissolved in ethanol, was added to the lipids before the removal of the organic solvent. Unilamellar liposomes were prepared as described above. After excitation at 350 nm, fluorescence emission of Laurdan was measured between 400 and 550 nm with the band-pass filters set to 2 nm. Generalized polarization (GP) values were then calculated with GP ¼ (I440  I490)/(I440 þ I490) (17).

Giant unilamellar vesicle preparation and microscopy Giant unilamellar vesicles (GUVs) were prepared by hydration of hybrid films of agarose and lipids (18). A 0.1 (w/w)% low-melting agarose solution (USB, Cleveland, OH) was prepared in pure water above the melting temperature. 20 mL of the agarose solution was deposited on a 10-mm cover glass and heated to evaporate the water. 3 mM lipid solution composed of SOPC with 16 mol% cholesterol and 7 mol% bSM and 0.1 mol% Liss Rhod phosphatidylethanolamine (PE) C18:1 in chloroform/methanol (2:1) were prepared, and 3 mL was spread on the agarose film. After at least 1 h of vacuum desiccation, the lipid film was hydrated with 100 mL 10 mM Hepes (pH 7.4) and 150 mM NaCl. Vesicle swelling was allowed for 1 h. The GUVs were removed from agarose and transferred for microscopic observations. After 3 h of settling, GUVs were examined with a 20 objective using a ZEISS Axio Observer.Z1 setup with Colibri 7-illumination module (ZEISS, Jena, Germany).

Circular dichroism spectroscopy Circular dichroism (CD) spectra were recorded at 25 C with step scanning speed using a Jasco J-815 spectropolarimeter with a bandwidth of 5 nm, data pitch of 1 nm, and a 0.1-cm path length quartz cells from Hellma (M€ ullheim, Germany). Unilamellar liposomes were prepared as described above at a (unlabeled) peptide/lipid ratio of 1:33 (i.e., 0.5 mM lipid and 15 mM peptide). The dried lipid/peptide film was hydrated with 10 mM phosphate buffer (pH 7.4). Each peptide CD spectrum was accumulated five times, and the buffer spectrum was subtracted.

Image processing with Chimera Molecular graphics and analyses performed with University of California, San Francisco Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (San Francisco, CA) (19).

RESULTS Glutamine 187 is key for the dimerization of the p24 TM helix The formation of p24 oligomers is not well understood yet, albeit an involvement of the TM helix has been suggested recently (3). Thus, to be able to study the (potential) impact of lipid binding on p24 TM helix dimerization, we first tested whether the isolated p24 TM helix has an intrinsic propensity to oligomerize within a biological membrane using the TOXCAT assay (12,20). In the TOXCAT assay, a TM helix of interest is genetically fused to the DNA-binding domain of the ToxR transcription activator of V. cholera, and oligomerization of the TM domains results in the formation of a dimeric DNA-binding domain that initiates expression of the cat reporter gene (12). In the end, a determined CAT activity of the reporter strains correlates with the oligomerization propensity of a given TM helix. The TOXCAT and related assays have been applied to numerous TM helices in the past 20 years and are well established in the field to determine the oligomerization propensities of TM helices (12,14,20–23). To identify residues crucially involved in the dimerization of the p24 TM helix, we mutated all residues of the TM helix and determined the CAT activities (Fig. 1, A and B) (i.e., the dimerization propensity of the mutated helices). As a control, we additionally determined the CAT activities in strains expressing the strongly dimerizing TM helix of the human GpA wt protein and the weakly dimerizing GpA G83I TM helix as well as in a strain carrying the empty expression plasmid (pcc). For all constructs, the protein expression level was tested (Fig. 1 C) as well as proper insertion of the chimeric protein into the E. coli inner membrane (i.e., localization of the DNA-binding domain within the bacterial cytoplasm) (Fig. S1). All chimeric proteins were expressed at comparable levels (Fig. 1 C) and with correct topology (Fig. S1), allowing a direct comparison of the determined CAT activities. The p24 wt TM helix showed a CAT activity similar to the activity determined in a strain expressing the GpA TM helix, and thus, the isolated p24 TM helix appears to have a strong intrinsic propensity to self-oligomerize. Whereas most of the mutated TM helices had an interaction propensity similar to the wt, the p24 TM mutants L180A and Q187A had a significantly decreased interaction propensity. Mutation of Leu180 resulted in an
40% decreased dimerization propensity, whereas replacement of Gln187 diminished dimerization to
10% of the wt level (Fig. 1 B). Thus,

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Pannwitt et al. FIGURE 1 Homodimerization of the p24 TM helix. (A) Amino acid sequences of the p24 and GpATM helices (bold) were analyzed in this study. The residues given in italics are present in the chemically synthesized peptides but not in the TOXCAT constructs. The underlined residues were mutated in the GpA G83I and p24 Q187A TM helices, respectively. (B) Homodimerization of wt and mutated p24 TM helices was determined using the TOXCAT assay. Each amino acid of the p24 TM region was mutated to Ala, the naturally occurring Ala residue to Leu. All measured CAT activities are given relative to the GpA wt activity (set as 1.0). wt represents p24 or GpA wt sequence, respectively; G83I represents GpA G83I mutated sequence; and () represents the empty plasmid pcc. Bars represent the mean 5 SD of results from at least five independent measurements. (C) Expression of the chimeric proteins in each strain studied with the TOXCAT assay was tested via Western blot analyses. Antibodies were directed against the maltose-binding protein domain. All proteins were expressed at a similar level. (D and E) The p24 Q187A TM helix shows reduced dimerization in liposomes composed of EPL (T ¼ 25 C). (D) Interaction of fluorescently labeled p24 wt and Q187A TM peptides was determined in unilamellar E. coli polar lipid liposomes via FRET measurements. Energy transfer from ATTO 499- to ATTO 590-labeled p24 Q187A peptides is reduced compared to the wt peptides. n ¼ 3 5 SD. (E) The linear dependence of the energy transfer (R2p24 wt ¼ 0.996, R2p24 Q187A ¼ 0.996) at increasing acceptor mole ratios demonstrates exclusive formation of helix dimers (25). n ¼ 3 5 SD.

Gln187 clearly is of special importance for the formation of p24 TM helix dimers. However, we intended to analyze the impact of cholesterol and SM binding on p24 TM helix dimerization at well-defined conditions, which would not be possible in cellular systems. Thus, we decided to perform measurements in model membranes that have distinct lipid compositions. Hence, we next tested dimerization of the isolated p24 TM helix in liposomes. For the FRET analyses, peptides corresponding to the p24 TM helix (Fig. 1 A) were chemically synthesized and labeled at their N-terminus with the fluorescent dyes ATTO 488 and ATTO 590, respectively. For experimental details, see Materials and Methods. To test dimerization of the p24 TM helix in lipid bilayers, the peptides were first analyzed after reconstitution into unilamellar liposomes produced of E. coli lipid extract. As can be seen in Fig. 1 D, the chemically synthesized p24 wt TM peptides strongly interacted in model membranes, confirming our in vivo observations (Fig. 1 B). Besides the wt sequence, we also analyzed the interaction of chemically synthesized p24 Q187A TM peptides in model membranes (Fig. 1 D). Our in vitro analyses nicely confirmed our in vivo data because the energy transfer (i.e., the stability of the TM dimer) was dramatically reduced when Gln187 was mutated (Fig. 1 D). However, although the dimerization propensity of p24 Q187A was
90% reduced in the TOXCAT assay (Fig. 1 A), it was ‘‘only’’
60% reduced in the in vitro assay in model membranes. Similar differences between in vivo and in vitro studies have already been observed with other proteins (24). We still had to rule out the possibility of chemically synthesized peptides unspecifically aggregating

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in vitro, which could have resulted in increased energy transfer signals. To test whether the p24 TM helix exclusively forms dimers, we performed FRET measurements at a constant total peptide and lipid concentrations, whereas the ratio of acceptor and donor peptides varied between 0.1 and 0.9. Only when the determined FRET efficiency linearly correlates to the mole fraction of the acceptor, the TM peptides exclusively form a dimer, as outlined in detail in (25,26). As can be seen in Fig. 1 E, the FRET efficiencies of both the p24 wt and Q187A peptides linearly correlate with the acceptor mole fraction, and thus, both peptides exclusively form dimers in model membranes. Together, the isolated p24 TM helix forms dimers in biological membranes as well as in model membranes, and Q187 is crucial for TM helix dimerization. Cholesterol does not directly affect p24 TM helix dimerization Based on computational predictions, it has been suggested that cholesterol specifically binds to the p24 TM helix (4). The cholesterol-binding motif has been predicted to involve the residues Tyr189 and Ile188 as well as Gln187, the residue identified in the TOXCAT assay as being crucial for p24 TM helix dimerization (Fig. 1 A). In fact, when we analyzed dimerization of wt p24 TM peptides in phosphatidylcholine (PC) membranes, we observed an increased dimerization propensity with increasing cholesterol content (Fig. 2 A), which might suggest that cholesterol binding to the p24 TM helix stabilizes the TM helix dimer. However, when we analyzed the dimerization propensity of the Q187A mutated p24 TM helix, we observed an essentially identical

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Lipid Binding Controls p24 Dimerization FIGURE 2 Cholesterol influences the rigidity of bilayers and indirectly affects dimerization of p24. (A) Energy transfer between labeled p24 wt (black), p24 Q187A (red), and GpA wt (gray) TM helices was determined in SOPC membranes containing different molar fractions of cholesterol (T ¼ 25 C). The dimerization propensity of all investigated TM peptides increases linearly with the cholesterol content. Data shown are the mean (5SD) of three independent measurements, whereby each value is the mean of three individual replicates. (B) The Laurdan GP value linearly increases with increasing cholesterol content, thus the addition of cholesterol rigidifies SOPC membranes (T ¼ 25 C). (C) Membrane lipid order directly affects the oligomerization of TM helices. Energy transfer between differentially labeled p24 wt TM helices was measured in SOPC liposomes at temperatures ranging from 5 to 25 C. The melting temperature of SOPC membranes is at around 5 C. Dimerization of p24 wt TM helix is enhanced when the membrane is in a gel phase. Data shown (B and C) are the mean 5 SD of three independent experiments.

trend (Fig. 2 A). Although the dimerization propensity of the mutant was generally lower, in line with the preceding in vivo and in vitro measurements (Fig. 1), the dimerization propensity still increased linearly with increasing cholesterol contents. Importantly, based on CD analyses, the a-helical content of both peptides is
70% (Fig. S2), and we observed the exclusive formation of dimeric structures (Fig. S3), ruling out the possibility of an increased energy transfer caused by a cholesterol-induced unspecific aggregation. However, we also observed a steady increase in dimerization of the unrelated GpA TM helix in liposomes containing increasing cholesterol concentrations, albeit GpA does not bind cholesterol (Fig. 2 A). When incorporated into a lipid bilayer, cholesterol significantly affects the structure of biological membranes (27), and this bulk membrane effect can indirectly affect dimerization of TM helices (16). Thus, we next determined the lipid order in our model membranes using the fluorescent probe Laurdan. In line with previous studies, the GP value, a measure for the membrane lipid order, increases linearly with the cholesterol content (Fig. 2 B). Thus, the increased dimerization propensities observed with the p24 wt and Q187A TM peptides most likely did not originate from the direct binding of cholesterol to the TM region but from a cholesterol-dependent alteration of the membrane structure, resulting in dimerization of the p24 TM helix. In line with this assumption, dimerization of the p24 TM helix is increased when a SOPC membrane is in the liquid-ordered phase (Fig. 2 C, T < 5 C) as compared to the liquid-disordered membrane phase (Fig. 2 C, T > 5 C), and thus, cholesterol-induced membrane lipid ordering, but not direct cholesterol binding, triggers dimerization of the p24 TM helix. Modulation of p24 dimerization by SM C18:0 depends on the lipid order As a direct binding of SM C18:0 to the p24 TM helix has recently been shown and an impact on p24 TM helix dimerization has been suggested (3), we next performed FRET experiments with the p24 wt TM helix in model membranes containing SM C18:0 (Fig. 3 A). Noteworthy, we used brain

SM (bSM), in which SM C18:0 is the major constituent (3). When p24 TM helix dimerization was analyzed in PC membranes containing increasing bSM concentrations, we did not observe any impact of bSM on the determined energy transfer (Fig. 3 A). As in vivo (i.e., in eukaryotic membranes), cholesterol and SM are present together, we next tested p24 TM wt helix dimerization in membranes containing both cholesterol and bSM. In our initial experiments, we used 7% bSM and 16% cholesterol, which mimics the lipid composition of Golgi membranes (3,28). Importantly, we specifically avoided the risk of lipid phase separation by using SOPC (i.e., a PC lipid with a rather high phase transition temperature), and at the lipid concentrations used in this study, lipid phase separation and the formation of segregated lipid domains was neither expected (29) nor observed (Fig. S4). To largely rule out the possibility that dimer formation is kinetically hindered in model membranes, we monitored dimer formation for 8 h (Fig. S5). At least within this time frame, dimerization did not change as for the p24 TM helices. The addition of 7% bSM to SOPC liposomes did not result in an altered p24 dimerization propensity (Fig. 3 B), whereas the addition of solely 16% cholesterol significantly enhanced p24 TM helix dimerization (Fig. 3 B). When both 7% bSM and 16% cholesterol were present, the observed dimerization propensity was increased compared to pure SOPC liposomes but equivalent to SOPC/ cholesterol liposomes (Fig. 3 B). However, the lipid order of liposomes containing both SM and cholesterol was somewhat increased compared to SOPC liposomes containing solely cholesterol (Fig. 3 C). Yet, the observed small changes in the lipid order (GP value) might not result in energy transfer efficiencies significantly differing from the p24 wt TM helix in SOPC plus cholesterol membranes (within the SD). However, we speculated that p24 TM helix dimerization induced by (cholesterol-induced) lipid ordering (compare Fig. 2, A and C) might be counteracted by the binding of SM 18:0 to the p24 TM helix. Hence, we next tested how changes in the lipid bilayer fluidity affect p24 TM helix interactions and probed dimerization of the p24 TM helix in the absence and presence of bSM

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FIGURE 3 SM binding affects dimerization of the p24 TM helix. (A) Brain SM (bSM) does not affect dimerization of the p24 wt TM helix when (SOPC) membranes are in the liquid-crystalline phase. Energy transfer between labeled p24 wt TM peptides (B) and GP values (C) was determined in pure SOPC liposomes or SOPC liposomes containing either 7 mol% bSM or 16 mol % cholesterol (chol) or both. (D and E) Energy transfer between labeled p24 wt TM peptides (black), p24 L185F TM peptides (gray) (D), and GP values (E) were determined in pure DOPC, SOPC, and DSPC liposomes, respectively, as well as in liposomes containing 9 or 18 mol% bSM. GP values are representatives of means 5 SD of three individual experiments, and energy transfer mean values were calculated of three mean values (n ¼ 3) of three individual experiments (T ¼ 25 C).

in PC lipids with increasing Tms, 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC; Tm ¼ 17 C, low Tm), SOPC (Tm ¼ 5 C, medium Tm), and 1,2-distearoyl-sn-glycero-3phosphocholine (DSPC; Tm ¼ 55 C, high Tm) membranes, respectively, at 25 C. Importantly, the PC acyl chain length was kept constant to avoid a significant impact of an altered bilayer thickness on p24 TM helix dimerization. Although dimerization of the p24 wt TM helix was identical in DOPC and SOPC bilayers (i.e., the membranes in the liquid-crystalline state) (Fig. 3, D and E), energy transfer was more than twofold enhanced in the gel-phase DSPC membranes (Fig. 3, D and E). The addition of bSM did neither change the lipid order of pure DOPC and SOPC membranes nor FRET between the labeled p24 TM helices (Fig. 3, D and E). Yet, the addition of bSM to the gel-phase DSPC membranes suppressed p24 wt dimerization. Thus, SM C18 appears to counteract dimerization of p24, induced by the membrane lipid order. To test whether specific binding of SM 18:0 to the p24 TM helix is crucial for the observed modulation of helix dimerization in gelphase membranes, we next analyzed the dimerization of the p24 L185F TM helix in SOPC membranes with increasing bSM content. Mutation of L185 to F has recently been shown to abolish specific binding of SM 18:0 to the p24 TM domain (3). As shown in Fig. 3 D, dimerization of the p24 TM helix was not affected anymore in DSPC membranes containing increasing amounts of bSM when SM 18.0 binding was abolished. Thus, specific binding of

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SM C18 to the p24 TM helix appears to counteract unspecific dimerization of p24, induced by bulk membrane effects.

DISCUSSION An LQ7 motif drives dimerization of the p24 TM helix Close packing, hydrogen bonding, Van der Waals interactions, and interactions of polar residues drive association of a-helices within lipid bilayers (30). In several cases, defined interaction motifs have been identified in recent years that mediate and stabilize TM helix oligomerization, resulting in the formation of well-defined TM structures (28,31–33). The currently best characterized interaction motif still is the GxxxG motif (34–38) and variations of it (i.e., small xxx small), but other motifs have also been described (31,33,39,40). Even though human TM sequences are relatively glycine rich and GxxxG motifs are highly abundant in TM proteins (36), GxxxG (-like) motifs are not per se crucial for self-association of TM a-helices (34,41). Although p24 TM regions contain a GxxxG-like motif (A182xxxG186), this motif is not conserved within the p24 protein family (Fig. 4 B), and our mutational analysis clearly shows that these residues are not crucial for interaction of the p24 TM helix (Fig. 1 B). In contrast, substitution of Gln187 resulted in significantly reduced

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Lipid Binding Controls p24 Dimerization FIGURE 4 Dimerization of and lipid binding to the p24 TM helix. (A) Model of the p24 TM helix dimer was calculated using PREDDIMER (45,46). Residues Gln187, Leu180, and Ser173 are at the dimerization interface. Highlighted are potential interhelical hydrogen bonds formed between the Gln187 side chain and backbone carbonyl oxygens and amino hydrogens of the adjacent helix. For clarity reasons, only the Gln187 residue of one helix is shown together with the interaction on the adjacent helix. Interacting amino acids are indicated. (B) Alignment of TM helix sequences of p24 family proteins of Homo sapiens and of other organisms is shown. Highlighted are residues involved in p24 TM helix dimerization (green) and residues of the (small)xxx(small) motif (gray). (C) Shown are amino acids involved in cholesterol and/or SM C18:0 binding as well as dimerization of the p24 TM helix. Residues (potentially) involved in lipid binding have been identified in (3) and (4), respectively.

interaction of the p24 TM helix. Importantly, a Gln residue at a homologous position is highly conserved in p24 proteins of different organisms (Fig. 4 B). Even though Gln is not highly abundant in membrane proteins (36), Gln residues tend to locate at conserved positions within TM helices in which they interact with cofactors, substrate, or polar residues on neighboring TM helices (42). In fact, polar interactions involving Gln residues can drive the formation of stable oligomeric TM structures (40,42–44), in line with our results (Fig. 1 B). Seven residues upstream of Gln187, and of homologous positions in other proteins of the p24 family, a Leu residue is strictly conserved (Fig. 4 B). Leu180 of the here analyzed p24 protein appears to further stabilize the association of two p24 helixes, but its impact on p24 TM helix dimerization is not as pronounced as Gln187 (Fig. 1 B). Yet, this residue is located at the same helix surface as Gln187 and, thus, likely is part of a p24 TM helix-helix interaction surface. In fact, the top-scoring structure of the dimeric p24 TM helix calculated using the program PREDDIMER (45,46) had an interaction surface that involves exactly these two residues (Fig. 4 A; Fig. S6). Noteworthy, Gln187was not involved in the dimerization in any of the remaining top-scoring predictions. Based on our experimental results, together with the predicted structure, the p24 TM helix likely forms a left-handed dimer with a crossing angle of 10 . In the projection of surface properties (molecular hydrophobicity potential and landscape), not only Gln187 and Leu180 but also Ser173 is located at the dimerization interface, leading to a rather polar dimerization interface (Fig. S6). Yet, favorable interactions with residues of the adjacent p24 TM helix are more pronounced for Gln187 and Leu180 than for Ser173 (Fig. 4 A), in line with the observation that the mutation of Ser173 did not affect p24 TM helix dimerization (Fig. 1 B). Although Leu180 likely mediates and stabilizes dimeriza-

tion via close packing, the Gln187 side chain can form numerous hydrogen bonds to backbone hydrogens and oxygens of the interacting helix (Fig. 4 A). In summary, p24 TM helix dimerization is mainly driven by the Leu180xxxxxx Gln187 (LQ7) sequence, leading to the rather small helix-crossing angle of 10 . Binding of SM C18:0, but not of cholesterol, to p24 affects TM helix dimerization An SM C18:0 binding motif has been identified in the p24 TM sequence, and cholesterol binding to the TM region has been suggested (3,4). The latter suggestion was tempting, as Gln187, which we have identified here as being key for TM helix dimerization (Fig. 1), has been suggested to be crucial for cholesterol binding (4). Thus, it was well possible that helix dimerization is connected to cholesterol binding, and cholesterol binding inhibits or enhances helix dimerization, as observed in the case of the amyloid precursor protein C99, for example (47). However, the results of our study essentially rule out the possibility of cholesterol directly influencing dimerization of the p24 TM helix. Although we observed an increased interaction propensity in cholesterol-containing membranes (Fig. 2 A), this was also observed when the Q187A-mutated p24 and the GpA wt TM helices were analyzed. Although both helices were not expected to bind cholesterol (anymore), the membrane lipid order (i.e., the membrane fluidity) is known to affect the dimerization of single-span TM proteins (16). Increasing the cholesterol content in model membranes visibly increased the lipid order (Fig. 2 B), and interaction of the p24 wt, Q187A, and GpA wt TM peptides linearly increased with an increasing lipid order (Fig. 2 A). Because Gln187 is clearly involved in helix dimerization (Fig. 1, B and D) and proposed to be crucial for cholesterol binding (4) and because dimerization of the p24 Q187A TM helix

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Pannwitt et al.

essentially increased in parallel to the wt helix in cholesterol-containing membranes, we in large part rule out the possibility that the p24 TM helix binds cholesterol. At least, any (potential) cholesterol binding does not have a significant impact on helix dimerization. In contrast to cholesterol, specific binding of SM C18:0 to the p24 TM region has recently been shown experimentally (3). The results presented in this study demonstrate that the presence of SM C18:0 decreases p24 TM helix dimerization in gel-phase or liquid-ordered lipid bilayers, whereas it does not affect dimerization in liquid-crystalline lipid bilayers, even in the presence of cholesterol (Fig. 3 B). This indicates that SM binding to the p24 TM helix can modulate helix dimerization under certain conditions. Importantly, the addition of bSM did not change the fluidity of DOPC, SOPC, or DSPC model membranes (Fig. 3 E). Although dimerization of the p24 TM helix in gel-phase DSPC liposomes was decreased after addition of bSM compared to fluid phase membranes, dimerization of the p24 L185F TM helix, which was supposed to not bind SM C18:0 anymore (3), was not altered (Fig. 3 D). Thus, specific binding of SM C18:0 reduces the stability of the p24 TM helix dimer, thereby counteracting the observed indirect effect of the increased lipid order on TM helix dimerization. At first view, our findings appear to contradict in part the recent conclusion that SM C18:0 binding enhances (rather than diminishes) dimerization of p24 proteins (3). However, in contrast to the study presented in (3), in this study, dimerization was assayed under equilibrium conditions. Additionally, here, we analyzed dimerization in PC liposomes with and without bSM, whereas in (3), dimerization of a p24 TM helix-containing fusion protein was determined and compared in PC liposomes containing either egg SM or bSM. Any (inhibiting) impact of egg SM on p24 TM helix dimerization was not considered. It has been suggested that SM C18:0 might affect p24 dimerization directly via inducing conformational changes upon binding and/or via inducing the accumulation of p24 proteins in SM C18:0-rich lipid domains (3). However, the question whether SM binds preferentially to monomeric or dimeric p24 or to both species likewise has not been experimentally addressed yet. However, because Q187 is crucial for p24 TM helix dimerization (this study) but not for SM 18:0 binding (3), it appears to be likely that SM binds better to monomeric p24 TM, at least in ordered bilayers. Conformational changes induced by SM 18:0-binding to the TM helix could thus lead to p24 monomerization in ordered bilayers, involving SM- and cholesterol-rich lipid domains, excluding COP I vesicle budding in these membrane areas (48).

to the ER, and the p24 protein shuttles in between the ER and the Golgi membrane. Yet, the cholesterol concentration of the ER (8%) and the Golgi (16%) differ substantially, which clearly affects the dimerization of p24 in the respective membrane system. However, although the ratio of monomeric versus dimeric p24 is similar in ER and Golgi membranes, the concentration of SM is also higher in the Golgi (8%) than in the ER (3%). Thus, based on our results, we propose that SM C18:0 binding to the p24 TM helix counteracts (indirect) cholesterol-induced dimerization of the p24 protein, especially in the Golgi, thereby ensuring comparable p24 dimerization propensities in internal membrane systems. As spatiotemporal control of lipid distribution and organization within defined membrane systems is a rather challenging task in living cells, we are confident that our findings will trigger supplementary and continuative in vivo studies. SUPPORTING MATERIAL Supporting Material can be found online at https://doi.org/10.1016/j.bpj. 2019.09.021.

AUTHOR CONTRIBUTIONS Conceptualization: S.P., M.S., and D.S.; methodology: S.P. and M.S.; validation: S.P. and M.S.; formal analysis: S.P., M.S., and D.S.; investigation: S.P. and M.S.; resources: D.S.; data curation: S.P. and M.S.; writing: S.P. and D.S.; visualization: S.P., M.S., and D.S.; supervision: D.S.; project administration: D.S.; and funding acquisition: D.S.

ACKNOWLEDGMENTS We thank Prof. Nadja Hellman for discussion and careful reading the manuscript and Hildegard Pearson for proofreading the manuscript. This work was supported by DynaMem (Rhineland-Palatinate, Germany).

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