Transitions of protein traffic from cardiac ER to junctional SR

• Published in: Journal of molecular and cellular cardiology 2015-04, Vol.81, p.34-45
• Main Authors: Sleiman, Naama H, McFarland, Timothy P, Jones, Larry R, Cala, Steven E
• Format: Article
• Language: English
• Subjects: Animals; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - metabolism; Cardiovascular; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell Nucleus - metabolism; Cell Nucleus - ultrastructure; Dogs; Gene Expression Regulation; Genes, Reporter; Junctin; Junctional sarcoplasmic reticulum; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Membrane Proteins - genetics; Membrane Proteins - metabolism; Microscopy, Fluorescence; Microtubules - metabolism; Microtubules - ultrastructure; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Muscle Proteins - genetics; Muscle Proteins - metabolism; Myocardium - cytology; Myocardium - metabolism; Myocytes, Cardiac - metabolism; Myocytes, Cardiac - ultrastructure; Protein Transport; Rats; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Rough ER; Sarcoplasmic Reticulum - classification; Sarcoplasmic Reticulum - metabolism; Sarcoplasmic Reticulum - ultrastructure; Signal Transduction; Smooth ER; Transgenes; Triadin
• ISSN: 0022-2828; 1095-8584
• DOI: 10.1016/j.yjmcc.2014.12.025

Abstract The junctional sarcoplasmic reticulum (jSR) is an important and unique ER subdomain in the adult myocyte that concentrates resident proteins to regulate Ca 2 + release. To investigate cellular mechanisms for sorting and trafficking proteins to jSR, we overexpressed canine forms of junctin (JCT) or triadin (TRD) in adult rat cardiomyocytes. Protein accumulation over time was visualized by confocal fluorescence microscopy using species–specific antibodies. Newly synthesized JCTdog and TRDdog appeared by 12–24 h as bright fluorescent puncta close to the nuclear surface, decreasing in intensity with increasing radial distance. With increasing time (24–48 h), fluorescent puncta appeared at further radial distances from the nuclear surface, eventually populating jSR similar to steady-state patterns. CSQ2-DsRed, a form of CSQ that polymerizes ectopically in rough ER, prevented anterograde traffic of newly made TRDdog and JCTdog , demonstrating common pathways of intracellular trafficking as well as in situ binding to CSQ2 in juxtanuclear rough ER. Reversal of CSQ-DsRed interactions occurred when a form of TRDdog was used in which CSQ2-binding sites are removed (del TRD). With increasing levels of expression, CSQ2-DsRed revealed a novel smooth ER network that surrounds nuclei and connects the nuclear axis. TRDdog was retained in smooth ER by binding to CSQ2-DsRed, but escaped to populate jSR puncta. TRDdog anddel TRD were therefore able to elucidate areas of ER–SR transition. High levels of CSQ2-DsRed in the ER led to loss of jSR puncta labeling, suggesting a plasticity of ER–SR transition sites. We propose a model of ER and SR protein traffic along microtubules, with prominent transverse/radial ER trafficking of JCT and TRD along Z-lines to populate jSR, and an abundant longitudinal/axial smooth ER between and encircling myonuclei, from which jSR proteins traffic.