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)P-2 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.

Rossi, D., Scarcella, A.M., Liguori, E., Lorenzini, S., Pierantozzi, E., Kutchukian, C., et al. (2019). Molecular determinants of homo- and heteromeric interactions of Junctophilin-1 at triads in adult skeletal muscle fibers. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 116(31), 15716-15724 [10.1073/pnas.1820980116].

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

Rossi, Daniela;Scarcella, Angela Maria;Liguori, Enea;Lorenzini, Stefania;Pierantozzi, Enrico;Sorrentino, Vincenzo
2019-01-01

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)P-2 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.
Rossi, D., Scarcella, A.M., Liguori, E., Lorenzini, S., Pierantozzi, E., Kutchukian, C., et al. (2019). Molecular determinants of homo- and heteromeric interactions of Junctophilin-1 at triads in adult skeletal muscle fibers. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 116(31), 15716-15724 [10.1073/pnas.1820980116].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11365/1079344