Assembly and structure of native PMCA-Neuroplastin/Basigin complexes

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Transient changes in the intracellular calcium (Ca2+) concentration, generally known as Ca2+ signaling, represents a prevalent mechanism to control a multitude of cellular processes and reaction pathways from transmitter release and regulation of enzymatic activities to excitation-transcription coupling, cell motility and neurite outgrowth. All these processes are switched on by influx of Ca2+ into the cytosol, and they are switched off by the concerted action of various Ca2+ transporters, most importantly Ca2+-ATPases (or pumps) that are located in the sarco/endoplasmic reticulum (SERCA) or the plasma membrane (PMCA). In recent proteomic analyses, we identified PMCAs in any type of cell as heteromeric complexes of ATPase subunits (PMCAs1-4) and the single-span membrane proteins Neuroplastin (NPTN) or Basigin (BASI). Co-assembly of the latter with the PMCA proteins is obligatory for both stabilization and trafficking of the complexes, as well as for effective substrate transport. In fact, binding of NPTN or BASI increases the rates of Ca2+-transport thus promoting Ca2+-clearing within tens of milliseconds rather than seconds, as previously assumed. The structural basis behind the assembly PMCAs with NPTN and BASI are currently unknown, as are the interaction partners of the PMCA- NPTN/ BASI core complexes in distinct tissues. This proposal aims at a comprehensive understanding of the assembly and structure of PMCA-NPTN/BASI complexes and their molecular appearance in native tissue(s). For this purpose we will pursue the following closely interrelated aims and experimental approaches/steps: (1) Elucidate 3D-structures of PMCA2-NPTN and PMCA4-BASI complexes, and of PMCA2 in auxiliary subunit-free ‘apo’ state by cryo-EM (Aim1A), (2) Measure ATP-consumption of purified PMCA2-NPTN/BASI complexes by a novel MS-based activity assay (Aim1B, (3) Unravel the interactome of native PMCA complexes (Aim2).