Secondary Proteins:
  1. mCherry,
  2. mCherry2
Add photostability measurements

Excerpts

This strongly suggests that the fluorescent protein tag caused clustering artifacts (Fig. 2a) and that ClpX and ClpP fluorescent protein fusions cannot be trusted for determining localization of native, untagged proteins. [...] These findings motivated us to evaluate other fluorescent proteins fused to ClpP or ClpX. We found that sfGFP, Venus, mCherry and mCherry2 all caused substantial foci formation, despite being monomers or very weak dimers when expressed alone. mKate2 and TagRFP-T caused intermediate clustering, whereas with mVenus and mYPet most of the fluorescence signal was spatially uniform, although we observed foci in a few cells. mTagBFP and mEos2 fusions resulted in a weak signal with infrequent dim foci. We detected no foci for photoswitchable (PS)-CFP2, reversible switchable (rs)FastLime (data not shown) and a mutant of sfGFP with a charge of −30 (GFP(−30)), but the signals were very dim. Finally, mGFPmut3, Dronpa and Dendra2 displayed an essentially uniform signal. Fluorescent protein fusions to ClpP generally caused more foci formation than fusions to ClpX, in particular for mYPet (Fig. 2d).

This strongly suggests that the fluorescent protein tag caused clustering artifacts (Fig. 2a) and that ClpX and ClpP fluorescent protein fusions cannot be trusted for determining localization of native, untagged proteins. [...] These findings motivated us to evaluate other fluorescent proteins fused to ClpP or ClpX. We found that sfGFP, Venus, mCherry and mCherry2 all caused substantial foci formation, despite being monomers or very weak dimers when expressed alone. mKate2 and TagRFP-T caused intermediate clustering, whereas with mVenus and mYPet most of the fluorescence signal was spatially uniform, although we observed foci in a few cells. mTagBFP and mEos2 fusions resulted in a weak signal with infrequent dim foci. We detected no foci for photoswitchable (PS)-CFP2, reversible switchable (rs)FastLime (data not shown) and a mutant of sfGFP with a charge of −30 (GFP(−30)), but the signals were very dim. Finally, mGFPmut3, Dronpa and Dendra2 displayed an essentially uniform signal. Fluorescent protein fusions to ClpP generally caused more foci formation than fusions to ClpX, in particular for mYPet (Fig. 2d).