Bioluminescence resonance energy transfer (BRET) betweenRenilla luciferase and yellow fluorescent protein has been adapted to serve as a real-time reporter on protein-protein interactions in live plant cells by using the Arabidopsis Constitutive photomorphogenesis 1 (COP1) protein as a model system. COP1 is a repressor of light signal transduction that functions as part of a nuclear E3 ubiquitin ligase. COP1 possesses a leucine-rich nuclear-exclusion signal that resides in a domain implicated in COP1 dimerization. BRET was applied in conjunction with site-directed mutagenesis to explore the respective contributions of the nuclear-exclusion and dimerization motifs to the regulation of COP1 activity in vivo. One specific mutant protein, COP1 L105A , showed increased nuclear accumulation but retained the ability to dimerize, as monitored by BRET, whereas other mutations inhibited both nuclear exclusion and COP1 dimerization. Mutant rescue and overexpression experiments indicated that nuclear exclusion of COP1 protein is a rate-limiting step in light signal transduction.dimerization ͉ photomorphogenesis ͉ nuclear export T he etiolation response of Arabidopsis seedlings germinating in darkness is mediated in part by the Constitutive photomorphogenesis 1 (COP1) protein, a repressor of light-regulated development. COP1 functions in the nucleus (1) by targeting light-regulatory transcription factors for ubiquitin-mediated degradation by the proteasome (2-5). COP1 possesses E3 ubiquitin ligase activity, presumably in conjunction with the COP1-interacting proteins CIP8 and SPA1 (4-7). Accordingly, cop1 mutant plants show transcriptional misregulation of numerous light-inducible genes in darkness (8).A single bipartite nuclear localization signal is responsible for COP1 nuclear localization and function (1, 9). Within the nucleus, COP1 localizes to subnuclear speckles (10), which also may contain ubiquitination targets and regulators of COP1 activity (4,11,12). However, COP1 is subject also to exclusion from the nucleus, mediated by a 110-residue domain referred to as a cytoplasmic localization signal (CLS), which overlaps a predicted ␣-helical coiled-coil (CC) region (9). The nuclear exclusion of a ␤-glucuronidase-COP1 fusion protein is enhanced by light (13-15). When fused to ␤-glucuronidase or GFP, COP1 in fact is predominantly cytoplasmic and accumulates in a cytoplasmic inclusion body.Testing the hypothesis that the nuclear exclusion of COP1 by the CLS does, in fact, down-regulate COP1 activity in Arabidopsis is not trivial, because the CLS overlaps the CC domain, which governs both COP1 dimerization (15) and targeting to nuclear speckles (10). Thus, three aspects of COP1 that are tightly linked to one another are nuclear localization and dimerization and their role in the function of COP1. To test the dimerization of COP1 after mutagenesis within the CLS, we decided to apply bioluminescence resonance energy transfer (BRET) (16). In a BRET experiment, two candidate interaction partners are tagged with the blue-lightem...

The ability of HDL to support macrophage cholesterol efflux is an integral part of its atheroprotective action. Augmenting this ability, especially when HDL cholesterol efflux capacity from macrophages is poor, represents a promising therapeutic strategy. One approach to enhancing macrophage cholesterol efflux is infusing blood with HDL mimics. Previously, we reported the synthesis of a functional mimic of HDL (fmHDL) that consists of a gold nanoparticle template, a phospholipid bilayer, and apo A-I. In this work, we characterize the ability of fmHDL to support the well-established pathways of cellular cholesterol efflux from model cell lines and primary macrophages. fmHDL received cell cholesterol by unmediated (aqueous) and ABCG1- and scavenger receptor class B type I (SR-BI)-mediated diffusion. Furthermore, the fmHDL holoparticle accepted cholesterol and phospholipid by the ABCA1 pathway. These results demonstrate that fmHDL supports all the cholesterol efflux pathways available to native HDL and thus, represents a promising infusible therapeutic for enhancing macrophage cholesterol efflux. fmHDL accepts cholesterol from cells by all known pathways of cholesterol efflux: unmediated, ABCG1- and SR-BI-mediated diffusion, and through ABCA1.

Nascent high-density lipoprotein (HDL) particles arise in different sizes. We have sought to uncover factors that control this size heterogeneity. Gel filtration, native PAGE, and protein cross-linking were used to analyze the size heterogeneity of nascent HDL produced by BHK-ABCA1, RAW 264.7, J774, and HepG2 cells under different levels of two factors considered as a ratio, the availability of apolipoprotein AI (apoAI) -accessible cell lipid, and concentration of extracellular lipid-free apoAI. Increases in the available cell lipid:apoAI ratio due to either elevated ATP-binding cassette transporter A1 (ABCA1) expression and activity or raised cell density (i.e., increasing numerator) shifted the production of nascent HDL from smaller particles with fewer apoAI molecules per particle and fewer molecules of choline-phospholipid and cholesterol per apoAI molecule to larger particles that contained more apoAI and more lipid per molecule of apoAI. A further shift to larger particles was observed in BHK-ABCA1 cells when the available cell lipid:apoAI ratio was raised still higher by decreasing the apoAI concentration (i.e., the denominator). These changes in nascent HDL biogenesis were reminiscent of the transition that occurs in the size composition of reconstituted HDL in response to an increasing initial lipid:apoAI molar ratio. Thus, the ratio of available cell lipid:apoAI is a fundamental cause of nascent HDL size heterogeneity, and rHDL formation is a good model of nascent HDL biogenesis.—Lyssenko, N. N., Nickel, M., Tang, C., Phillips, M. C. Factors controlling nascent high-density lipoprotein particle heterogeneity: ATP-binding cassette transporter A1 activity and cell lipid and apolipoprotein AI availability.