Trafficking of Phospholamban and SERCA2a Follows the Nuclear Envelope-to-SR Along T-Tubules (NEST) Pathway Common to Junctional Sarcoplasmic Reticulum (SR) Proteins

Abstract

The pathways and mechanisms by which phospholamban (PLB) and SERCA2a proteins traffic to SR sarcomeres within cardiomyocytes (CMs) remain unknown. Applying a similar approach used to identify trafficking pathways for the junctional SR proteins triadin and junctin, we analyzed confocal immunofluorescence images of adult rat CMs 24-72hrs post infection with adenoviruses encoding dog isoforms of PLB (dPLB) or dSERCA2a. Newly-made dPLB or dSERCA2a proteins in CMs were specifically detected using species-specific monoclonal antibodies 1F1 or 3F1, respectively. At 24hrs, dSERCA2a (3F1 signals) were present in the nuclear envelope (NE), where discrete puncta also appeared, likely reflecting rough ER translocons producing presorted sites of localized biosynthesis. dSERCA2a protein distributed along transverse tracks emanating from translocons, with radially decreased levels. At the 3rd puncta away from the NE, 3F1 fluorescence intensity reduced to 52±12% (15 CMs, 4 rats) of that in the NE, indicating movement of dSERCA2a protein. In contrast, dPLB, detected by 1F1, was distributed throughout the NE (16 CMs), without detectable transverse/radial trajectory even at 24hrs post infection. By 48-72hrs post infection, fluorescence signals for both dPLB and dSERCA2a became visible in puncta further away from the NE, but still aligned with T-tubules; a pattern previously found for junctin and triadin. Thus, newly-synthesized PLB and SERCA2a travel along the same pathway used to deliver proteins to junctional SR. This unique protein trafficking pathway is termed the Nuclear Envelope-to-SR along T-tubules (NEST) pathway. After biosynthesis, but before trafficking to SR, PLB is preferentially retained in the NE at levels greater than SERCA2a, consistent with our recent report of enhanced steady-state PLB localization to the NE in CMs. The NEST pathway may be a common secretory pathway for cardiac SR protein trafficking.