Geranylgeranylated SCF regulates selective outer mitochondrial membrane proteostasis and function.

Compartment-specific cellular membrane protein turnover is not well understood. We show that FBXO10, the interchangeable component of the cullin-RING-ligase 1 complex, undergoes lipid modification with geranylgeranyl isoprenoid at cysteine953, facilitating its dynamic trafficking to the outer mitochondrial membrane (OMM). FBXO10 polypeptide lacks a canonical mitochondrial targeting sequence (MTS); instead, its geranylgeranylation at C953 and interaction with two cytosolic factors, cytosolic factor-like δ subunit of type 6 phosphodiesterase (PDE6δ; a prenyl-group-binding protein) and heat shock protein 90 (HSP90; a chaperone), orchestrate specific OMM targeting of prenyl-FBXO10.

Geranylgeranylated-SCF Regulates Selective Outer Mitochondrial Membrane Proteostasis and Function.

E3-ubiquitin ligases (E3s) are main components of the ubiquitin-proteasome system (UPS), as they determine substrate specificity in response to internal and external cues to regulate protein homeostasis. However, the regulation of membrane protein ubiquitination by E3s within distinct cell membrane compartments or organelles is not well understood. We show that FBXO10, the interchangeable component of the SKP1/CUL1/F-box ubiquitin ligase complex (SCF-E3), undergoes lipid-modification with geranylgeranyl isoprenoid at Cysteine953 (C953), facilitating its dynamic trafficking to the outer mitochondrial membrane (OMM).

Detection of membrane-anchoring lipid modifications of proteins in cells by radioactive metabolic labeling.

Prenylation and palmitoylation are two major lipid modifications of cellular proteins that anchor proteins to cell membranes. Here, we present a protocol for detecting these modifications in cellular proteins by radioactive metabolic labeling. We describe steps for metabolic labeling of cells, cell harvesting for carrying out immunoprecipitations, subjecting immunocomplexes to SDS-PAGE, and transferring them to polyvinylidine flouride (PVDF) membranes.

Palmitoylation and PDE6δ regulate membrane-compartment-specific substrate ubiquitylation and degradation.

Substrate degradation by the ubiquitin proteasome system (UPS) in specific membrane compartments remains elusive. Here, we show that the interplay of two lipid modifications and PDE6δ regulates compartmental substrate targeting via the SCF. FBXL2 is palmitoylated in a prenylation-dependent manner on cysteines 417 and 419 juxtaposed to the CaaX motif.

Ubiquitin proteasome system in immune regulation and therapeutics.

The ubiquitin proteasome system (UPS) is a proteolytic machinery for the degradation of protein substrates that are post-translationally conjugated with ubiquitin polymers through the enzymatic action of ubiquitin ligases, in a process termed ubiquitylation. Ubiquitylation of substrates precedes their proteolysis via proteasomes, a hierarchical feature of UPS. E3-ubiquitin ligases recruit protein substrates providing specificity for ubiquitylation.