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. 2006 Dec 14;103(52):19689–19694. doi: 10.1073/pnas.0609502103

Fig. 1.

Fig. 1.

Efficient mitochondrial targeting of multidomain ZFP proteins requires a combination of a specific MTS and NES. (A) ZFPs containing four fingers (F1–F4) were selected from two libraries (L1 and L2) to bind targets in mtDNA (SI Fig. 5). All ZFPs (with or without a GGG linker) were closely related. Sequences of recognition helices (positions −1 to 6) are shown with DNA-contacting amino acids in boldface. ZFPs were fused to N-terminal MTSs of the F1β subunit of the human mitochondrial ATP synthase (F-ZFP) or subunit VIII of human cytochrome c oxidase (C8-ZFP) in the presence or absence of C-terminal GFP (C8-ZFP-GFP), and their localization was assessed as exclusively nuclear (N), exclusively mitochondrial (M), or mixed with either predominantly mitochondrial (M/N) or predominantly nuclear (N/M). (B) The localization of a particular ZFP, ZFP30, was analyzed further in the context of different MTSs with or without additional sequences such as GFP (image 2) or 3′ UTR of the mRNA for F1β subunit of human ATP synthase (32) (image 8). In addition to C8 (images 1 and 2) and F (images 7 and 8), we tested the MTS from the subunit 6 of ATP synthase from Chlamydomonas reinhardtii (R) (image 6) and MTSs from the following zinc finger proteins: MP42 from Trypanosoma brucei (T1) (image 3), MP63 from T. brucei (T2) (image 4), and 7b from Leishmania tarentolae (T3) (image 5). In the merged immunofluorescence images mitochondria are stained in red and ZFPs are labeled in green, and partially mitochondrial localization of F-ZFP30 is marked by arrows (image 7). (C and D) A number of F-ZFPs were fused with the C-terminal NES (F-ZFP–NES) and tested for their ability to enter mitochondria when attached to additional domains including GFP (F-GFP–ZFP–NES, 48 kDa), a catalytic domain of the hDNMT3a methylase (F-ZFP–meth–NES, 61 kDa), or both (F-GFP-ZFP–meth–NES, 86 kDa). Intracellular localization of individual proteins was assessed and presented (C) and additionally, for clone ZFP30, illustrated by images in D (abbreviations as in A and B).