Abstract
Green fluorescent protein (GFP)-tagging is the prevalent strategy to monitor protein dynamics in living cells. However, the consequences of appending the bulky GFP moiety to the protein of interest are rarely investigated. Here, using a powerful combination of quantitative fluorescence spectroscopic and imaging techniques, we have examined the oligomerization dynamics of the GFP-tagged mitochondrial fission GTPase dynamin-related protein 1 (Drp1) both in vitro and in vivo. We find that GFP-tagged Drp1 exhibits impaired oligomerization equilibria in solution that corresponds to a greatly diminished cooperative GTPase activity in comparison to native Drp1. Consequently, GFP-tagged Drp1 constitutes aberrantly stable, GTP-resistant supramolecular assemblies both in vitro and in vivo, neither of which reflects a more dynamic native Drp1 oligomerization state. Indeed, GFP-tagged Drp1 is detected more frequently per unit length over mitochondria in Drp1-null mouse embryonic fibroblasts (MEFs) compared to wild-type (wt) MEFs, indicating that the drastically reduced GTP turnover restricts oligomer disassembly from the mitochondrial surface relative to mixed oligomers comprising native and GFP-tagged Drp1. Yet, GFP-tagged Drp1 retains the capacity to mediate membrane constriction in vitro and mitochondrial division in vivo. These findings suggest that instead of robust assembly-disassembly dynamics, persistent Drp1 higher-order oligomerization over membranes is sufficient for mitochondrial fission.
Highlights
Green fluorescent protein (GFP)-tagging is the prevalent strategy to monitor protein dynamics in living cells
In many instances, GFP-tagging has been shown to generate unstable fusion products[4,5], cause aberrant protein localization[6], promote aggregation[7], prevent assembly[8], and/or perturb protein function in more than subtle ways[9,10,11,12]. Such scrutiny has never been imposed on GFP-tagged dynamin-related protein 1 (Drp1), a longstudied yet controversial mechanoenzymatic GTPase recruited from the cytosol to the mitochondrial surface to mediate mitochondrial d ivision[13,14]
N-terminally tagged monomeric enhanced GFP (mEGFP)-Drp[1] and C-terminally tagged Drp1-mEGFP were overexpressed in E. coli for recombinant protein production and biochemical and biophysical characterization in vitro, and in mouse embryonic fibroblasts (MEFs) for examining Drp[1] quaternary structure, oligomerization dynamics, and function in vivo
Summary
Green fluorescent protein (GFP)-tagging is the prevalent strategy to monitor protein dynamics in living cells. GFP-tagged Drp[1] retains the capacity to mediate membrane constriction in vitro and mitochondrial division in vivo These findings suggest that instead of robust assembly-disassembly dynamics, persistent Drp[1] higher-order oligomerization over membranes is sufficient for mitochondrial fission. Modified BODIPY-FL (BODIPY)-labeled Drp[1] efficiently catalyzed Drp1-wt-like constriction of relatively flat membranes, but not fission, under comparable conditions[17] These conflicting results suggested that the various fluorescent tags adorning Drp[1] might not be passive constituents as originally assumed but instead actively influence Drp[1] structure and/or function. We show that GFP-tagging at either end disrupts Drp[1] oligomerization equilibria both in solution and on membranes by favoring Drp[1] higher-order self-assembly and limiting GTP hydrolysis-mediated oligomer disassembly. Our results caution against the interpretation of results from disparately labeled Drp[1] constructs without appropriate controls, and biochemical and biophysical information in place
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