Small Heat-Shock Proteins Regulate Membrane Lipid Polymorphism
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Small heat-shock proteins regulate membrane lipid polymorphism Nelly M. Tsvetkova*†, Ibolya Horva´ th‡, Zsolt To¨ ro¨ k‡, Willem F. Wolkers*, Zsolt Balogi‡, Natalia Shigapova‡, Lois M. Crowe*, Fern Tablin§, Elizabeth Vierling¶, John H. Crowe*, and La´ szlo´ Vi´gh‡ *Section of Molecular and Cellular Biology, §Department of Anatomy, Physiology, and Cell Biology, University of California, Davis, CA 95616; ¶Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ 85721; and ‡Institute of Biochemistry, Biological Research Center, 6701, Szeged, Hungary Communicated by Tibor Farkas, Hungarian Academy of Sciences, Szeged, Hungary, August 5, 2002 (received for review March 7, 2002) Thermal stress in living cells produces multiple changes that ulti- referred to as the ␣-crystallin domain and have monomeric mately affect membrane structure and function. We report that two masses of 16–40 kDa (6). Many sHsps have been shown to be members of the family of small heat-shock proteins (sHsp) (␣-crys- efficient at binding denatured proteins, and current models tallin and Synechocystis HSP17) have stabilizing effects on model propose that sHsps function as molecular chaperones preventing membranes formed of synthetic and cyanobacterial lipids. In anionic irreversible protein aggregation (7, 8). Increased expression of membranes of dimyristoylphosphatidylglycerol and dimyristoylphos- sHsps can increase tolerance to a variety of stresses, such as heat, phatidylserine, both HSP17 and ␣-crystallin strongly stabilize the salt, drugs, and oxidants (9). liquid-crystalline state. Evidence from infrared spectroscopy indicates ␣-Crystallin, a major component of the lens in vertebrate eyes, is that lipid͞sHsp interactions are mediated by the polar headgroup a member of the sHsp family and is composed of two highly region and that the proteins strongly affect the hydrophobic core. In homologous subunits, ␣A and ␣B (10). Initially thought to be membranes composed of the nonbilayer lipid dielaidoylphosphati- lens-specific proteins, both ␣A- and ␣B-crystallin have been iden- dylethanolamine, both HSP17 and ␣-crystallin inhibit the formation of tified also in heart, liver, and brain (11). Despite numerous reports, inverted hexagonal structure and stabilize the bilayer liquid-crystal- the biological role of ␣-crystallin is not yet firmly established. It is line state, suggesting that sHsps can modulate membrane lipid thought to inhibit stress-induced aggregation in vitro, suggesting a polymorphism. In membranes composed of monogalactosyldiacyl- potential role in maintenance of lens transparency (8, 12). ␣-Crys- glycerol and phosphatidylglycerol (both enriched with unsaturated tallin has been shown to bind to ocular plasma membranes and ␣ fatty acids) isolated from Synechocystis thylakoids, HSP17 and -crys- synthetic phospholipid vesicles (13–18), but the mechanism of tallin increase the molecular order in the fluid-like state. The data interaction is not yet characterized, and the role these interactions ͞ show that the nature of sHsp membrane interactions depends on the may play in lens biology is not clear. A recent report (19) suggests lipid composition and extent of lipid unsaturation, and that sHsps that increased membrane association of ␣-crystallin is a critical can regulate membrane fluidity. We infer from these results that event in the pathogenesis of cataracts. the association between sHsps and membranes may constitute A correlation between the level of sHsps and thermotolerance a general mechanism that preserves membrane integrity during in a cyanobacterium Synechocystis PCC 6803 was published thermal fluctuations. recently (20). Inactivation of the single Synechocystis sHsp gene, hsp17, reduced the photosynthetic oxygen evolution in heat- he physical state of the membrane lipids is a central feature of stressed cells. It was shown that part of the protein is associated Tcurrent models of membrane organization (1). Although bio- with thylakoid membranes and, by increasing the membrane logical membranes maintain an overall bilayer structure under physical order, antagonizes the heat-induced increase in fluidity, physiological conditions, they contain substantial amounts of lipids thus protecting the membranes from thermal damage (21). like phosphatidylethanolamine (PE) and monogalactosyldiacylglyc- The objective of this work is to reveal the mode of interactions erol (MGDG) with strong preference to organize into nonbilayer between sHsps and membranes composed of synthetic and structures, such as inverted hexagonal phase (HII). Eukaryotic cyanobacterial lipids. We used two members of the sHsp family, membranes are usually highly enriched in PE, whereas MGDG is ␣-crystallin and HSP17, which share low sequence homology the major lipid component of thylakoid membranes in plant cells. [22% in the C-terminal extension, 24% in the ␣-crystallin A dominant structural feature of these lipids is a small polar domain, and no homology in the N-terminal region (22)] but headgroup compared with large acyl chains, resulting in a conical affect lipid membranes in a similar way. The study documents a shape (2). This shape can induce local high-membrane curvature, novel feature of ␣-crystallin and HSP17; namely, they stabilize resulting in the formation of inverted micelles. It is known that the bilayer liquid-crystalline phase relative to the inverted membrane physical properties are maintained well within the hexagonal phase in lipids with high propensity for nonbilayer lamellar liquid-crystalline state but with a constant potential for structures (PE, MGDG). We found that sHsps strongly modu- forming nonbilayer structures. Numerous data suggest that the late the membrane properties of anionic phospholipids, suggest- formation of such structures is precisely regulated, and that they are ing a protection by sHsps at both thermal extremes. This novel of fundamental importance for fusion, endocytosis, signaling, and property of sHsps implies local (domain-restricted) membrane for the activity of certain membrane-bound enzymes (2–4). How- functions, provided by the discrete localization of membrane ever, nonbilayer structures can be induced by environmental stress, lipids. The data also suggest that the association between sHsps such as elevated temperatures, thereby producing transient mem- brane defects and affecting permeability (5). Therefore, during thermal stress, it is essential to prevent their formation and to Abbreviations: sHsp, small heat-shock protein; DMPG, dimyristoylphosphatidylglycerol; preserve membrane integrity. DMPS, dimyristoylphosphatidylserine; DEPE, dielaidoylphosphatidylethanolamine; Virtually all organisms respond to high temperature by syn- MGDG, monogalactosyldiacylglycerol; PG, phosphatidylglycerol; HII, inverted hexagonal structure; Tm, phase transition from gel to liquid-crystalline state; Thex, phase transition thesizing heat-shock proteins, including the small heat-shock from lamellar to hexagonal phase; FTIR, Fourier transform infrared spectroscopy; DSC, proteins (sHsps). The sHsps are a diverse group of stress proteins differential scanning calorimetry; Tes, N-[tris(hydroxymethyl)methyl]-2-aminoethanesul- that are sequence related to the vertebrate lens ␣-crystallins. fonic acid. They are defined by a conserved C-terminal domain of Ϸ100 aa †To whom correspondence should be addressed. E-mail: [email protected]. 13504–13509 ͉ PNAS ͉ October 15, 2002 ͉ vol. 99 ͉ no. 21 www.pnas.org͞cgi͞doi͞10.1073͞pnas.192468399 Downloaded by guest on September 29, 2021 and membranes may constitute a general mechanism, which (HP3396A Hewlett–Packard) on a SP2330 column (Supelco, protects membranes against thermal fluctuations. Bellefonte, PA) as in ref. 23. Materials and Methods Results Materials. Synthetic dimyristoylphosphatidylcholine, dimyris- Molecular interactions between proteins and lipid membranes toylphosphatidylglycerol (sodium salt) (DMPG), dimyristoylphos- can be studied by monitoring discrete frequency ranges in the phatidylserine (sodium salt) (DMPS), and dielaidoylphosphati- FTIR spectrum. In this work, we observed changes in the dylethanolamine (DEPE) were purchased from Avanti Polar Lipids vibrational frequency of the methylene symmetric stretching Ϫ1 and used without further purification. Synechocystis PCC 6803 was mode ( CH2) at 2,850 cm , which reflects the membrane ␦ grown as in ref. 23, and Synechocystis thylakoids were isolated as in fluidity, and the methylene scissoring band ( CH2) at 1,468 ref. 23. MGDG and phosphatidylglycerol (PG) were isolated from cmϪ1, which is diagnostic for the lateral packing in the depth of Synechocystis thylakoids by using chloroform͞methanol extraction the hydrophobic core (28, 29). We also followed changes in the Ϫ (2:1) and purified by two-dimensional thin-layer chromatography as lipid phosphate asymmetric stretching band ( PO2 ) at 1,220 ␣ Ϫ1 Ϫ in ref. 24. -Crystallin was purchased from StressGen Biotechnol- cm and the serine carboxylate stretching band ( CO2 ) at 1,420 ogies (Victoria, Canada). HSP17 was expressed in Escherichia coli cmϪ1, which report on the immediate environment of the lipid (strain GI724, Invitrogen) carrying the plasmid pAL-hsp17, and headgroup region (30). purified as in ref. 25. Influence of ␣-Crystallin and HSP17 on Bilayer-Forming Lipids. In a Sample Preparation. Liposomes were prepared by drying aliquots of previous study, we showed that HSP17 did not affect the gel to the lipid under an N stream, followed by at least2honalyophilizer.