This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.D-AKAP1 is a PKA anchoring protein that targets PKA to mitochondria or ER. After defining the N-terminal mitochondrial targeting motif, the mitochondrial targeting forms of D-AKAP1 are being characterized for their localization and function using electron microscopy and electron tomography. It has been known for the past decade that compartmentalization of PKA can occur through association with A kinase anchoring proteins (AKAPs). This association ensures specificity in signal transduction by placing kinases close to their appropriate substrates where they can swiftly respond to second messengers. Another advantage of compartmentation to specific subcellular locations is to restrict accessibility to certain substrates. For PKA binding, it was thought for a long time that only RII subunits bind to AKAPs. However, a new class of AKAPs was discovered when two dual-specific AKAPs (D-AKAP1 and D-AKAP2) were cloned using a fusion protein of RI as the bait in a yeast two-hybrid screen. Therefore, the contrasted distribution pattern between RI and RII may in part be due to selective association with different compartmentalized pools of AKAPs. Our goal in using the NCMIR is to obtain a high-resolution 3D map of D-AKAP1 (and related proteins) labeling of mitochondria to determine if this anchoring protein is uniformly distributed on the outer membrane, or if it is localized. If localized, does it associate with any identifiable features of mitochondria, such as contact sites, constriction sites of buds, extensions of the outer membrane leading to autophagosomes, or crista junctions?
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