Molecular determinants of homo- and heteromeric interactions of Junctophilin-1 at triads in adult skeletal muscle fibers

Abstract

In adult skeletal muscles, 2 junctophilin isoforms (JPH1 and JPH2) tether the sarcoplasmic reticulum (SR) to transverse tubule (T-tubule) membranes, generating stable membrane contact sites known as triads. JPHs are anchored to the membrane of the SR by a C-terminal transmembrane domain (TMD) and bind the T-tubule membrane through their cytosolic N-terminal region, which contains 8 lipid-binding (MORN) motifs. By combining expression of GFP-JPH1 deletion mutants in skeletal muscle fibers with in vitro biochemical experiments, we investigated the molecular determinants of JPH1 recruitment at triads in adult skeletal muscle fibers. We found that MORN motifs bind PI(4,5)P2 in the sarcolemma, but do not mediate the selective localization of JPH1 at the T-tubule compartment of triads. On the contrary, fusion proteins containing only the TMD of JPH1 were able to localize at the junctional SR compartment of the triad. Bimolecular fluorescence complementation experiments indicated that the TMD of JPH1 can form dimers, suggesting that the observed localization at triads may result from dimerization with the TMDs of resident JPH1. A second domain, capable of mediating homo- and heterodimeric interactions between JPH1 and JPH2 was identified in the cytosolic region. FRAP experiments revealed that removal of either one of these 2 domains in JPH1 decreases the association of the resulting mutant proteins with triads. Altogether, these results suggest that the ability to establish homo- and heterodimeric interactions with resident JPHs may support the recruitment and stability of newly synthesized JPHs at triads in adult skeletal muscle fibers.

Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats

“…While there is abundant evidence that junctophilins associate with the plasma membrane, and that the MORN repeat-containing region is likely to mediate this, there is to the authors’ knowledge no paper demonstrating that the junctophilin MORN repeats directly interact with lipids (Takeshima et al, 2000, Munro, Jayasinghe et al, 2016, Woo, Srikanth et al, 2016, Perni, Lavorato et al, 2017, Jayasinghe, Clowsley et al, 2018, Nakada et al, 2018, Rossi et al, 2019). Despite this, the evidence that the MORN repeat-containing region mediates plasma membrane targeting has been repeatedly cited as evidence that MORN repeats directly bind lipids.…”

“…MORN repeats are generally assumed to be lipid-binding modules, but direct evidence for this function is actually lacking. In junctophilins, there is good evidence that the N-terminal region containing the MORN repeats mediates plasma membrane targeting (Takeshima et al, 2000, Nakada, Kashihara et al, 2018, Rossi, Scarcella et al, 2019). It has not been demonstrated whether the MORN repeats or the other sequences in the N-terminal region are responsible for this however, or if this targeting is due to protein-lipid or protein-protein interactions.…”

Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats

“…TbMORN1 truncations were generated using this construct as a template by ligase-independent cloning [34]. The sequences for TbMORN1 (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) and TbMORN1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) were additionally ligated into the pCoofy12 vector encoding a 3C protease-cleavable N-terminal Twin-Strep-tag [35] by sequence and ligation-independent cloning [36]. Mutagenesis constructs were generated by standard methods using the pre-existing pCoofy12_TbMORN1(2-15) construct as the template [37].…”

“…(B) TbMORN1 dimerises via its C-terminus. SEC-MALS profiles of TbMORN1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15), TbMORN1 (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15), and TbMORN1 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). Schematics are shown underneath.…”

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“…Despite their evolutionary distance, they share high (57% and 54%, respectively) sequence identity with TbMORN1 (S9A and S9B Fig). CD measurements of purified recombinant protein indicated that TgMORN1, TgMORN1(7-15), PfMORN1, PfMORN1 (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15), and PfMORN1 (7)(8)(9)(10)(11)(12)(13)(14)(15) all had an overall β-structure (S9C and S9D Fig). This agrees with bioinformatic predictions and is consistent with the data obtained for TbMORN1 (S1E Fig).…”