Identification and Characterization of Unique Proline-rich Peptides Binding to the Mitochondrial Fission Protein hFis1

Abstract

Mammalian mitochondrial fission requires at least two proteins, hFis1 and the dynamin-like GTPase DLP1/Drp1. The mitochondrial protein hFis1 is anchored at the outer membrane by a C-terminal transmembrane domain. The cytosolic domain of hFis1 contains six ␣ helices [␣1-␣6] out of which [␣2-␣5] form tetratricopeptide repeat (TPR)-like motifs. DLP1 and possibly other proteins are thought to interact with the hFis1 TPR region during the fission process. It has also been suggested that the ␣1-helix regulates protein-protein interactions at the TPR. We performed random peptide phage display screening using the hFis1[␣2-␣6] as the target and identified ten different peptide sequences. Phage ELISA using mutant hFis1 indicates that the peptide binding requires the ␣2 and ␣3 helices and the intact TPR structure. Competition experiments and surface plasmon resonance analyses confirmed that a subset of free peptides enriched with proline residues directly bind to the target. Two of these peptides bind to the ␣1-containing intact cytosolic domain of hFis1 with decreased affinity. Peptide microinjection into cells abolished the mitochondrial swelling induced by overexpression of ␣1-deleted hFis1, and significantly decreased cytochrome c release from mitochondria upon apoptotic induction. Our data demonstrate that hFis1 can bind to multiple amino acid sequences selectively, and that the TPR constitutes the main binding region of hFis1, providing a first insight into the hFis1 TPR as a potential therapeutic target.Mitochondria are dynamic organelles that undergo constant shape changes through processes including fission and fusion of the organelle tubules (1). Mitochondrial fission and fusion are evolutionarily conserved processes, suggesting their importance in basic cell function. It is presumed that fission and fusion of mitochondria allow the mixing of the mitochondrial genome as well as other mitochondrial components such as the respiratory chain complexes to maintain adequate organelle function (2-5). Mitochondrial dynamics is also required for the proper distribution of this organelle within cells especially in neurons (6 -8) and to ensure correct inheritance of this organelle to daughter cells during cell division (9). Defects in the fission or fusion machinery result in human disease conditions, such as subtypes of Charcot-Marie-Tooth disease and autosomal dominant optic atrophy, indicating their functional importance (10 -13). In addition, abnormal phenotypes of mitochondrial morphology have been reported in several neurodegenerative diseases such as Alzheimer, Parkinson, and Huntington diseases, and recent studies indicate the direct involvement of mitochondrial fission/fusion in these diseases (14 -18).The two major processes that govern mitochondrial morphology are fission and fusion of the organelle. Studies on mitochondrial fusion in mammals have shown that dynamin-related large GTPases, mitofusin (Mfn) and OPA1, participate in the fusion of outer and inner mitochondrial membranes, respectively (19 -24). Recent ...

The 1.75 Å resolution structure of fission protein Fis1 fromSaccharomyces cerevisiaereveals elusive interactions of the autoinhibitory domain

“…sufficient for ligand binding. This has recently been shown to be true for human Fis1ÁTM, which also contains only two TPR motifs (Serasinghe et al, 2010).…”

Cloning and characterization of a novel 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Salvia miltiorrhiza involved in diterpenoid tanshinone accumulation

“…The motifs rich in arginines (RRR) are considered to be important for endoplasmic reticulum retention (Boulaflous et al, 2009). On the other hand, proline residues are known to strongly modify the secondary structure of proteins (Serasinghe et al, 2010). Plant HMGR has been shown to insert in vitro into the membrane of microsomal vesicles, but the final in vivo subcellular localization remains controversial.…”

PEX11 proteins attract Mff and hFis1 to coordinate peroxisomal fission

“…Two prolines in the identified proline-rich motif, P165/167, resemble a motif, 'PLP', recently identified as binding sequence for hFis1 (Serasinghe et al, 2010), a factor of the peroxisomal fission machinery already shown to interact with the PEX11 proteins (Kobayashi et al, 2007;Koch et al, 2010). To decipher whether this tripeptide is involved in the regulation of PEX11c, we chose to study the effect of its deletion from the PEX11c sequence in vivo (Fig.…”