Folding Latency of Fluorescent Proteins Affects the Mitochondrial Localization of Fusion Proteins

Abstract

The discovery of fluorescent proteins (FPs) has revolutionized cell biology. The fusion of targeting sequences to FPs enables the investigation of cellular organelles and their dynamics; however, occasionally, such fluorescent fusion proteins (FFPs) exhibit behavior different from that of the native proteins. Here, we constructed a color pallet comprising different organelle markers and found that FFPs targeted to the mitochondria were mislocalized when fused to certain types of FPs. Such FPs included several variants of Aequorea victoria green FP (avGFP) and a monomeric variant of the red FP. Because the FFPs that are mislocalized include FPs with faster maturing or folding mutations, the increase in the maturation rate is likely to prevent their expected localization. Indeed, when we reintroduced amino acid substitutions so that the FP sequences were equivalent to that of wild-type avGFP, FFP localization to the mitochondria was significantly enhanced. Moreover, similar amino acid substitutions improved the localization of mitochondria-targeted pHluorin, which is a pHsensitive variant of GFP, and its capability to monitor pH changes in the mitochondrial matrix. Our findings demonstrate the importance of selecting FPs that maximize FFP function.

Interaction Between Phosphoinositide 3-Kinase and the VDAC2 Channel Tethers Endosomes to Mitochondria and Promotes Endosome Maturation

“…The expression vectors pCAGGS-VN-H-Ras-WT, pCXN2-FLAG-PI3K(p110γ)-RBD-VC, pFX-SECFP, pFX-ECFP-RAPEL, pFX-mito-SECFP, and pFX-mito-EGFP were described previously (Fujioka et al, 2018, 2019; Kashiwagi et al, 2019; Tsutsumi et al, 2009). The vector pCMV-TagRFP-EEA1 was obtained from Addgene (Cambridge, MA, USA).…”

“…The coding sequence of PI3K was cleaved out of pCXN2-FLAG-PI3K-RBD-VC by digestion with XhoI and NotI and was subcloned into the EcoRI and BglII sites of pFXII-V5-Rab5 to obtain pFXII-V5-PI3K. The coding sequence of A. thaliana CIB1 was amplified from pCIB1(deltaNLS)-pmGFP (Addgene) by PCR with the primer pair CIB1_F and CIB1_R and was then subcloned into the BamHI and BglII sites of pFX-TOM20-mCherry or pFX-TOM20-iRFP (Kashiwagi et al, 2019) to obtain pFX-TOM20-mCherry-CIB1 and pFX-TOM20-iRFP-CIB1, respectively. The cDNA for A. thaliana CRY2 was released from pCX4puro-CRY2-cRaf (kindly provided by K. Aoki, National Institutes for Basic Biology, Okazaki, Japan) by digestion with EcoRI and XhoI and was subcloned into the corresponding sites of pFX-FYVE to obtain pFX-CRY2-FYVE.…”

“…To reveal the molecular mechanism by which the Ras-PI3K complex is translocated from the plasma membrane to endosomes in a RAPEL-dependent manner (Fujioka et al, 2019), we screened for RAPEL binding proteins with the use of a yeast two-hybrid assay based on the RBD of PI3K (PI3K-RBD) as the bait ( Figure S1A). We identified six candidate proteins (Table S1), among which we focused on the mitochondrial pore protein VDAC2 because we were able to confirm its interaction with RAPEL in mammalian cells by immunoprecipitation analysis (Figure 1A).…”

“…Analysis of the endosomal localization of the Ras-PI3K complex was performed as described previously (Fujioka et al, 2019). In brief, confocal images of cells expressing Venus…”

“…We have previously shown that endosomal localization of a complex formed by the small GTPase Ras and PI3K promotes clathrin-independent endocytosis and endosome maturation (Fujioka et al, 2011). In addition, a 28-amino acid sequence at the NH2-terminal region of the Ras binding domain (RBD) of PI3K was found to be required for such endosomal localization and was thus designated RAPEL (Ras-PI3K endosomal localization) (Fujioka et al, 2019).…”