A Minimal Peptide Sequence That Targets Fluorescent and Functional Proteins into the Mitochondrial Intermembrane Space

Abstract

Protein-based fluorescent and functional probes are widely used for real-time visualization, purification, and regulation of a variety of biological molecules. The protein-based probes can generally be targeted into subcellular compartments of eukaryotic cells by a particular short peptide sequence. Little is known, however, about the sequence that targets probes into the mitochondrial intermembrane space (IMS). To identify the IMS-targeting sequence, we developed a simple genetic screening method to discriminate the proteins localized in the IMS from those in the mitochondrial matrix, thereby revealing the minimum requisite sequence for the IMS targeting. An IMS-localized protein, Smac/DIABLO, was randomly mutated, and the mitochondrial localization of each mutant was analyzed. We found that the four residues of Ala-Val-Pro-Ile are required for IMS localization, and a sequence of these four residues fused with matrix-targeting signals is sufficient for targeting the Smac/DIABLO into the IMS. The sequence was shown to readily direct three dissimilar proteins of interest to the IMS, which will open avenues to elucidating the functions of the IMS in live cells.

CHCM1/CHCHD6, Novel Mitochondrial Protein Linked to Regulation of Mitofilin and Mitochondrial Cristae Morphology

“…Smac was also detected in the M fraction but not in the OM or IM fraction (Fig. 3A), which may be consistent with recently reported findings that modified Smac localized to the matrix (40). Mitofilin is a known IM protein (41,42), and, as expected, it was detected predominantly in the IM fraction but not in the OM, IMS, and M fractions (Fig.…”

Mitochondria in lung biology and pathology: more than just a powerhouse

“…This enzyme functions as a monomer, whereas other H 2 O 2 scavengers only function as multimers. Sabharwal et al targeted its expression to the IMS using the NH 2 -terminal sequence that directs the SMAC/Diablo protein to that compartment (66) and confirmed correct localization using immunogold electron microscopy (79). When PRDX5 was expressed in the IMS of PASMC, it attenuated the increase in ROS in the IMS and the cytosol during hypoxia.…”

Plasmids for variable expression of proteins targeted to the mitochondrial matrix or intermembrane space

“…The 59-residue, IMSdirecting leader is that derived from the human SMAC/Diablo protein, as previously described. 25,26 We also included in our constructs the HA epitope (YPYDVPDYA), between the leader sequences and proteins of interest (see Fig. 1).…”