Vol. 50 No. 2/2003 337–365 QUARTERLY Review The structure of the Ca2+-ATPase of sarcoplasmic reticulum. Anthony N. Martonosi1 and Slawomir Pikula2½ 1State University of New York, Upstate Medical University, College of Graduate Studies, Syracuse, New York, U.S.A.; 2M. Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warszawa, Poland Received: 30 May, 2003; accepted: 02 June, 2003 Key words: calcium homeostasis, sarcoplasmic reticulum, calcium pump, calcium transport, excitation-contraction coupling, skeletal and cardiac muscles In this article the morphology of sarcoplasmic reticulum, classification of Ca2+-ATPase (SERCA) isoenzymes presented in this membrane system, as well as their topology will be reviewed. The focus is on the structure and interactions of Ca2+-ATPase determined by electron and X-ray crystallography, lamellar X-ray and neutron diffraction analysis of the profile structure of Ca2+-ATPase in sarcoplasmic reticulum multilayers. In addition, targeting of the Ca2+-ATPase to the sarcoplasmic reticulum is discussed. The classical studies of Heilbrunn and 1964) on the Ca2+ regulation of muscle con- Wierczinsky (1947), Ebashi (1961; 1962), traction by the sarcoplasmic reticulum (SR) Hasselbach (1961; 1963) and Weber (1959; set the stage for the exploration of the unique .This work was supported in part by grant No. 3 P04A 007 22 from the State Committee for Scientific Re- search (KBN, Poland). ½ Corresponding author: Slawomir Pikula, Department of Cellular Biochemistry, M. Nencki Institute of Experimental Biology, L. Pasteura 3, 02-093 Warszawa, Poland; tel.: (48 22) 659 8571 (ext. 347); fax: (48 22) 822 5342; e-mail: [email protected] Abbreviations: AMPPCP, 5¢-[beta,gamma-methylene]triphosphate; AMPPNP, adenosine 5¢-(beta,gamma-imino)triphosphate; BiP, immunoglobulin binding protein; caged ATP, P3-1(2-nitro)phenylethyladenosine-5¢-triphosphate; DHPR, dihydropyridine receptor; ER, endoplasmic reticulum; FITC, fluorescein-5¢-isothiocyanate; PLN, phospholamban; PMCA, plasma membrane Ca2+-ATPase; RyR, ryanodine receptor; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; SR, sarcoplasmic reticulum; TG, thapsigargin; TNP-AMP, 2¢,3¢-O-(2,4,6-trinitrophenyl)AMP; 8-azido-TNP-ATP, 8-azido-2¢,3¢-O-(2,4,6-trinitrophenyl)ATP; V, vanadate; T-tubules, transverse tubules. 338 A.N. Martonosi and S. Pikula 2003 role of calcium in the regulation of a wide Mitchell et al., 1988). Among the SERCA range of metabolic processes and for the eluci- Ca2+-ATPase isoenzymes, the SERCA1 and dation of mechanisms by which cytoplasmic SERCA2a Ca2+ transport ATPase isoforms and intraorganellar Ca2+ concentrations are are evenly distributed throughout the free SR controlled in living cells. and most of the lateral sac, but are apparently excluded from the junctional SR. The SERCA2b isoform may be preferentially local- THE MORPHOLOGY OF ized near the T-SR junction. Detailed classifi- SARCOPLASMIC RETICULUM cation of SERCA isoforms is described in the next paragraph. The SR of skeletal muscle consists of two morphologically and functionally distinct re- gions: the junctional SR and the free SR (for CLASSIFICATION OF Ca2+-ATPase review see Peachey & Franzini-Armstrong, ISOENZYMES 1983). The junctional SR membrane contains the ryanodine receptor (RyR) Ca2+ channel The determination of the amino-acid se- with feetlike projections on its cytoplasmic quences of the sarcoplasmic reticulum Ca2+- surface that interact with the dihydropyridine ATPase (MacLennan et al., 1985), and of the receptor (DHPR) particles contained in the closely related Na+,K+-ATPase (Kawakami et junctional region of the T-tubules, forming the al., 1985; Shull et al., 1985) has opened a new T-SR (triad or dyad) junction. The T-SR junc- era in the analysis of ion transport mecha- tion is involved in the transmission of the ex- nisms. Since 1985 several large families of citatory stimulus from the surface membrane structurally related ion transport enzymes to the SR, causing Ca2+ release from the were discovered that are the products of dif- sarcoplasmic reticulum (Martonosi & Pikula, ferent genes. Within each family several iso- 2003). The free SR contains the Ca2+ trans- enzymes may be produced from a single gene port ATPase as its principal membrane com- product by alternative splicing. Our discus- ponent. It is usually divided into the lateral sion will concentrate on the Ca2+ transport sacs (cisternae), and the longitudinal tubules, ATPases that occur in the SR of muscle cells which differ in protein composition. The lat- of diverse fiber types and in the endoplasmic eral sacs contain electron-dense material in reticulum (ER) of nonmuscle cells. their lumen, which is attributed to the The sarco/endoplasmic reticulum Ca2+- lumenal Ca2+-binding proteins of SR (calse- ATPases of mammalian tissues can be divided questrin, calreticulin, high affinity Ca2+-bind- structurally into 3 main groups (SERCA1–3) ing proteins, etc.) that may serve as storage representing the products of different genes. sites for the accumulated Ca2+. The slender The SERCA1 gene (ATP2A1) produces two longitudinal tubules connect the lateral sacs isoforms of the Ca2+-ATPase, that are derived through the center of the sarcomere and by alternative splicing of the primary gene across the Z line; they contain little or no product (MacLennan et al., 1985; Brandl et al., calsequestrin. 1986). SERCA1a denotes the Ca2+-ATPase of Differential and sucrose gradient centri- adult fast-twitch skeletal muscle with glycine fugation permits the separation of the various at its C-terminus in the rabbit (Brandl et al., membrane elements into vesicular fractions 1987; Korczak et al., 1988), and alanine in the enriched in T-tubules, lateral sacs, and longi- chicken (Ohnoki & Martonosi, 1980; Karin et tudinal tubules, which differ in Ca2+ transport al., 1989). The C terminus of the lobster en- activity and protein composition (Caswell et zyme is apparently blocked (Ohnoki & al., 1988; Chu et al., 1988; Costello et al., 1988; Martonosi, 1980). SERCA1b is the alterna- Vol. 50 Calcium pump of sarcoplasmic reticulum 339 tively spliced neonatal form of SERCA1, in a sorting signal for retention of the enzyme in which the glycine at the C-terminus is re- the endoplasmic reticulum (Burk et al., 1989). placed by the alternative sequence -Asp-Pro- Sequences of SERCA type Ca2+-ATPases Glu-Asp-Glu-Arg-Arg-Lys (Brandl et al., 1986; were also obtained from Plasmodium yoelii 1987). The gene encoding SERCA1 is on hu- (Murakami et al., 1990), Artemia (Palmero et man chromosome 16 (MacLennan et al., al., 1989), and Drosophila (Magyar et al., 1987). A selective defect in its expression is 1995). These enzymes are similar in size to the cause of some forms of Brody’s disease SERCA type Ca2+-ATPases from mammalian (MacLennan, 2000). muscles, but based on their N- and C-terminal The SERCA2 gene (ATP2A2) also produces sequences they represent a distinct group. In at least two isoforms that are tissue specific. spite of the wide phylogenetic variations be- SERCA2a is the principal form of the tween them, they all share a common N-ter- Ca2+-ATPase in adult slow-twitch skeletal and minal sequence (Met-Glu-Asp) that differs cardiac muscles and in neonatal skeletal mus- from mammalian enzymes. cles (Barndl et al., 1986; MacLennan et al., The molecular masses of all SERCA type 1987; Lytton et al., 1988; 1992; Wu et al., Ca2+ transport ATPases of muscle are close to 1995; Verboomen et al., 1995). It is also ex- 110 kDa. Their N-terminal sequences are simi- pressed at much lower levels in nonmuscle lar: Met-Glu-X(Ala, Asn, Glu, Asp)-X (Ala, Gly, cells (Wu et al., 1995). Its C-terminus is Ile). The Met-Glu-X-X sequence serves as sig- Pro-Ala-Ile-Leu-Glu. SERCA2b is an alterna- nal for the acetylation of N-terminal tively spliced product of the same gene methionine both in soluble and in membrane (Gunteski-Humblin et al., 1988; Lytton et al., proteins (Tong, 1977; 1980). 1989). It is located primarily in nonmuscle tis- sues and in smooth muscles, where it serves as the major intracellular Ca2+ pump. THE PREDICTED TOPOLOGY OF SERCA2b is characterized by a long Ca2+-ATPases C-terminal extension of 50 amino acid resi- dues ending in Trp-Ser (Gunteski-Humblin et Combining structural and biochemical infor- al., 1988; Lytton et al., 1988; 1989). The gene mation, MacLennan and his colleagues con- for both forms of SERCA2 is located on hu- structed a hypothetical model of the tertiary man chromosome 12 (MacLennan et al., structure of Ca2+-ATPase (MacLennan et al., 1987). Its expression is not affected in Brody’s 1985; 1997) that had interesting mechanistic disease, suggesting that the two major forms implications (Fig. 1), and was largely con- of SR Ca2+-ATPases are independently regu- firmed by recent structural data (MacLennan lated. & Green, 2000; Toyoshima et al., 2000; Green SERCA3 is encoded by the ATP2A3 gene et al., 2002; Toyoshima & Nomura, 2002; (Dode et al., 1996). It is broadly distributed in Toyoshima et al., 2003). The structure was di- skeletal muscle, heart, uterus, and in a variety vided into three major parts, designated as of nonmuscle cells (Burk et al., 1989; Wuytack the cytoplasmic headpiece, the stalk domain et al., 1995; Dode et al., 1996; Kovacs et al., and the transmembrane domain. Only short 2001). The mRNA levels are particularly high loops were assumed to be exposed on the in intestine, lung and spleen, while it is very luminal side of the membrane. low in liver, testes, kidney and pancreas. In More than half of the total mass of the the muscle tissue SERCA3 may be confined ATPase molecule is exposed on the cytoplas- primarily to nonmuscle cells (endothelial mic surface of the membrane, forming the cy- cells, etc.). The C-terminus of SERCA3 is toplasmic head piece (Fig. 1). The headpiece Asp-Gly-Lys-Lys-Asp-Leu-Lys; it may serve as contains six subdomains: the N-terminal re- 340 A.N.