The role of junctophilin proteins in cellular function

Figures

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  FIGURE 1.. Predicted subcellular organization of junctophilin (JPH) within the junctional membrane complex consisting of the plasma membrane (PM) and endoplasmic/sarcoplasmic reticulum (ER/SR). JPH2 attaches to the PM by means of 2 sets of membrane occupation and recognition nexus (MORN) domains (green), connected by a junctional domain (J). The divergent (D) region with low homology among JPH isoforms. The COOH-terminal tail of JPH2 anchors into the endo/sarcoplasmic reticulum (ER/SR) membrane using a transmembrane domain, TA, tail anchor.

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  FIGURE 2.. Localization of junctophilin (JPH) genes on chromosomes. Ideograms of chromosomes (Chr.) 8, 20, 16, and 14 (p and q regions), showing the JPH1, JPH2, JPH3, and JPH4 gene locations, respectively. Cytogenic bands are showing in accordance with the International System for Cytogenetic Nomenclature (15). Idiograms were generated using files posted on the website of the Department of Pathology of the University of Washington (16).

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  FIGURE 3.. Gene and protein diagram of junctophilin (JPH) isoforms. Diagram shows gene diagram including exons and introns in correlation with the protein topology for the 4 JPH isoforms expressed in humans. The membrane occupation and recognition nexus (MORN) domains are shown in green, while the other domains are marked. The exons (E) are shown in pink. JPH, junctophilin; biNLS, biphasic nuclear localization signal; mNLS, monopartite nuclear localization signal; UTR, untranslated region; TM, transmembrane domain.

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  FIGURE 4.. Phylogenetic tree of junctophilin (JPH) isoforms. The phylogenetic tree was created using 57 JPH isoforms from 22 different species that were selected with different times of the last common ancestor. Protein sequences were aligned using MUSCLE in Mega-X using default settings, a distance matrix was generated, and the phylogenetic tree was generated using the neighbor joining method. Clades containing each of the isoforms are indicated. The scale bar represents the length of each branch as the average number of amino acid substitutions per site (per Mega-X).

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  FIGURE 5.. Evolutionary trace mapping of human junctophilin 2 (JPH2). Human JPH isoforms were aligned using the MUSCLE function in Mega-X using default settings. The universal evolutionary trace was generated using the Evolutionary Trace viewer created by Dr. O. Lichtarge (44). The human JPH2 sequence is shown as the reference sequence; the output was color coded based on the evolutionary trace scores (rvET) from the RANKS files. Lower scores (blue shaded squares) represent higher importance; higher scores represent a lower importance of amino acids (red shaded squares). Bolded amino acids are conserved across isoforms. Functional domains are underlined. MORN, membrane occupation and recognition nexus; NLS, nuclear localization signal.

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  FIGURE 6.. Evolutionary conservation of the junctophilin-2 (JHP2) membrane occupation and recognition nexus (MORN)-1 domain. Homologues of the Homo sapiens JPH2 protein were obtained using protein BLAST (see accession numbers). The selected top hit from each species with different evolutionary distances from humans were aligned using the MUSCLE feature in Mega-X. A distance matrix of the aligned sequences was generated using default settings. Estimated evolutionary distance was obtained from TimeTree (30). The colors represent the amino acid types; mya, millions of years ago.

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  FIGURE 7.. Phylogenetic tree of membrane occupation and recognition nexus (MORN) repeat containing protein families. The phylogenetic tree was constructed using 238 amino acid sequences from a range of proteins found in eukaryotes, bacteria, and archaea. The clustering of this tree revealed 8 distinct families of protein containing MORN repeat-containing proteins: the junctophilins (JPH), alsins (ALS), radial spoke-head homology (RSPH) proteins, four MORN-containing families (MORN1-MORN4), and an ankyrin repeat and MYND domain-containing protein 1-like (ANKMY-L) family, along with another less clearly defined group of proteins. The tree is to scale, with branch lengths corresponding to amino acid substitutions per site, calculated using the average pathway method. Reproduced with permission from Mackrill and Shiels (29).

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  FIGURE 8.. Junctophilin 2 (JPH2) 3-dimensional structural prediction model. JPH2 template-based tertiary structure modeling by RaptorX visualizing the backbone fold and atomic structure. Labels indicate major JPH2 domains and regions. Inset: magnification (dashed boxes) visualizing the COOH-terminal (C) transmembrane domain (TMD) α-helix of the JPH2 tail anchor in maximal side projection. N, NH2 terminal. Modified JPH2 model based on the same protein sequence from Gross et al. (60) using Molekel software. The color code for the atom structure is red for oxygen (O), blue for nitrogen (N), yellow for sulfur (S), and gray for carbon (C).

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  FIGURE 9.. Membrane topology, biogenesis, and endo/sarcoplasmic reticulum (ER/SR) membrane insertion of tail-anchored (TA) proteins via the guided entry of TA proteins (GET) pathway. A: topology comparison of the single-pass type II transmembrane protein integrin-α1 (PDB 2L8S) versus the type IV TA protein phospholamban (PDB 2HYN). B: space filling JPH2 3-dimensional (3-D) structure atomic prediction. Wild-type junctophilin 2 (JPH2) template-based tertiary structure modeling by RaptorX and Molekel. The ER/SR membrane leaflets’ approximate positions are indicated behind the TA COOH-terminal (C) domain (orange lines). N, NH2 terminal. Modified JPH2 model based on the same protein sequence from Gross et al. (60) using Molekel software. Molekel is an open-source 3-dimensional molecular visualization package for analyzing the results of computational chemistry packages (61). C: the transmembrane domain provides the moderately to strongly hydrophobic physicochemical properties, targeting TA proteins to the ER/SR organelle (62). D: at the end of ribosomal biogenesis the nascent polypeptide hydrophobic COOH-terminal TA (cyan) faces the challenging aqueous cytosolic environment. SRP, signal…

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  FIGURE 10.. Conserved 23-amino acid tandem membrane occupation and recognition nexus (MORN) repeat atomic structures and predicted junctophilin 2 (JPH2) β-strand architecture. A and B: Domain depiction and crystal structure of the NH2-terminal deletion proteins Trypanosoma brucei TbMORN1(7–15) and Toxoplasma gondii TgMORN1(7-15) each forming tail-to-tail homodimers. Amino acid numbers and NH2(N)/COOH (C) termini are indicated on top. The crystal structure is shown both from the side and 90° rotated as indicated. Major dimensions are indicated (double arrows). Each truncated protomer contains 9 MORN repeats of which the 3 COOH-terminal repeats additionally provide the antiparallel tail-to-tail interactions. The secondary structure consists exclusively of antiparallel β-strands and peripheral loops. TbMORN1(7–15) and TgMORN1(7–15) exhibit the same number of MORN repeats and structural configurations. Modified with permission from Sajko et al. (81). C: consensus MORN repeat sequence of TbMORN(7–15) revised according to its crystal structure. While repeats 7–15 exist in the crystal structure, the deleted repeats 1–6 are inferred. Blue color intensities indicate the conservation of sequence…

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  FIGURE 11.. Subcellular junctophilin 2 (JPH2) clustering in atrial and ventricular cardiomyocytes. A: mouse atrial cardiomyocyte confocal section overview (top) and magnified image region indicated by yellow brackets (bottom). Magnification showing the variable subcellular signal qualities of immunostained JPH2 clusters. Larger JPH2 cluster signals (red) marked by white arrowheads are mainly located at deep intracellular axial tubule structures labeled with caveolin-3 (green). Less intense JPH2 signals apparently localize to transverse striations devoid of Cav3 signals. Scale bar = 10 μm. Modified with permission from Brandenburg et al. (82). B: confocal 3-dimensional z-stack projection overview (top) and magnification (bottom) showing intense JPH2 cluster signals (green) mainly deep inside a mouse atrial cardiomyocyte, occasionally intersecting ryanodine receptor 2 (RyR2) channel clusters (red) in transversal striations evident by colocalized signals (yellow). Scale bar = 10 μm. Modified with permission from Brandenburg et al. (82). C: high-power magnification of rat ventricular myocyte. Exchange-PAINT quantitative superresolution imaging of a single cluster of RyR2 channels (red),…

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  FIGURE 12.. Subcellular ryanodine receptor 2 (RyR2) clustering in ventricular control and transgenic heart sections from adult mice. A-C: superresolution images showing immunolabeled RyR2 channel clusters in left-ventricular myocardial sections at transverse striations. A: junctophilin 2 (JPH2) knockdown (JPH2-KD). B: control wild-type heart section. C: JPH2 overexpression (JPH2-OE). Magnified views of singular clusters are shown in the insets. Scale bars = 4 µm in main panels; scale bars = 0.5 µm in insets indicated by white rectangles. D: RyR2 cluster size. E: number of RyR2 clusters. Mouse strains are indicated by color. Control, n = 11 cells, 2 animals; JPH2-KD, n = 12 cells, 2 animals; JPH2-OE, n = 9 cells, 2 animals. Data are displayed as means ± SE. ***P < 0.001 (Kruskal-Wallis two-sided test). Modified with permission from Munro et al. (74).