Identification of a novel 45 kDa protein (JP-45) from rabbit sarcoplasmic-reticulum junctional-face membrane

Abstract

Using a biochemical/immunological approach to analyse the protein constituents of skeletal-muscle junctional-face membrane (JFM), we identified a 45kDa protein. Its N-terminal amino acid was blocked, but the amino acid sequence obtained from several peptides after proteolytic treatment did not significantly match that of any protein present in the SwissProt and NCBI (National Center for Biotechnology Information) databases. We synthesized a peptide whose sequence matched that of one of the peptides obtained after CNBr cleavage of the 45kDa protein; the peptide was conjugated to a carrier and used to raise antibodies. The antiserum was used to study in more detail the biochemical characteristics of the novel 45kDa protein. Analysis of the proteins present in different subcellular membrane fractions show that the novel 45kDa polypeptide: (i) is an integral membrane constituent present both in neonatal and adult skeletal-muscle sarcoplasmic reticulum; (ii) is selectively localized in the JFM; (iii) is not present in microsomes obtained from rabbit heart, liver or kidney. Immunoprecitation with anti-(45kDa protein) antibody indicates that the 45kDa protein is part of a complex which can be phosphorylated in vitro by the catalytic subunit of protein kinase A.

The junctional SR protein JP-45 affects the functional expression of the voltage-dependent Ca2+ channel Cav1.1

“…In the past few years a number of investigators have begun to define the major and minor structural components of the junctional face membrane (Ito et al, 2001;Takeshima et al, 2000). In previous studies (Zorzato et al, 2000;Anderson et al, 2003), we identified and characterized -at the biochemical and molecular level -JP-45, an integral membrane protein constituent of the SR junctional face membrane in skeletal muscle. We also showed that JP-45 colocalizes with the RyR Ca 2+ -release channel and interacts with Ca v 1.1 and the luminal Ca 2+ -binding protein calsequestrin (Anderson et al, 2003).…”

“…Besides the RyR, however, the junctional face membrane contains numerous other proteins that, because of their anatomical location, are deemed to be involved in E-C coupling (Zhang et al, 1997;Costello et al, 1986;Zorzato et al, 2000). In the past few years a number of investigators have begun to define the major and minor structural components of the junctional face membrane (Ito et al, 2001;Takeshima et al, 2000).…”

Loss of skeletal muscle strength by ablation of the sarcoplasmic reticulum protein JP45

“…In addition to the calcium-release channel (RyR), the SR and T tubule membranes also have a variety of proteins that, because of their subcellular location, are thought to contribute to ECC (23,24). They are involved in (i) calcium storage (calsequestrin) (25), (ii) signal transduction (triadin TD, junctin JC, mitsugumin-29, JP45) (23,24,26,27), (iii) calcium homeostasis (Ca 2ϩ pump, Na/Ca exchanger) (28), and (iv) maintenance of SR spatial organization and integrity (junctophilin) (29).…”

“…They are involved in (i) calcium storage (calsequestrin) (25), (ii) signal transduction (triadin TD, junctin JC, mitsugumin-29, JP45) (23,24,26,27), (iii) calcium homeostasis (Ca 2ϩ pump, Na/Ca exchanger) (28), and (iv) maintenance of SR spatial organization and integrity (junctophilin) (29). Our previous studies (24,30) demonstrated that JP45, an integral protein constituent of the skeletal muscle SR junctional face membrane, interacts with Ca v 1.1 and the luminal calcium-binding protein calsequestrin (30,31). Ca v 1.1 and JP45 form a complex that is down-regulated during aging and may contribute to decayed muscle strength and physical disability in the elderly (31,32).…”

Increased Ca2+ storage capacity of the skeletal muscle sarcoplasmic reticulum of transgenic mice over‐expressing membrane bound calcium binding protein junctate

“…Biochemical data indicate that in addition to RyRs, the JFM contains several protein constituents, including triadin, calsequestrin, junctin, histidine rich Ca 2þ binding protein (HRC), mitsugumin-29, junctophilin, 90 kDa protein or JP-45 (MacLennan and Wong, 1971;Hofmann et al, 1989;Caswell et al, 1991;Jones et al, 1995;Takeshima et al, 1998Takeshima et al, , 2000Froemming et al, 1999;Zorzato et al, 2000;Anderson et al, 2003). However, the JFM is also endowed with numerous other less abundant proteins having a molecular mass ranging between 20 and 120 kDa (Costello et al, 1986), whose identity and functional role are still obscure.…”