Rix is mediated by a DYm-dependent mechanism (Fig. 6D). The residual mitochondrial signal can be explained by mitochondrial import prior to the CCCP treatment. Results describing the SR3029 localization and processing of constructs carrying an altered version of the NTS in HEK293T cells are similar to those obtained for D. discoideum. Constructs NTS DRS and NTS 105A are targeted to mitochondria and proteolytically processed (Fig. S3). Multiple bands were observed on immunoblots when lysates from HEK293T cells expressing these proteins were analysed (Fig. S3E). This might be due to the combined 25033180 effects of MPP and MIP or differences in specificity compared to their D. discoideum counterparts. The minimal construct (NTS DI2) is properly processed and targeted to mitochondria in HEK293T cells. Processing of DI2 in HEK 293T cells appears 
to be more efficient than in D. discoideum and gives similar products. Deletion of the R-recognition site or introduction of mutation R105A reduces the targeting efficiency of NTS DI2 (Fig. S4A ). Compared to the situation in D. discoideum, NTS DI2 DRS and NTS DI2 R105A are more completely processed in HEK 293T cells (Fig. S4D). However, we cannot exclude that the unspecific action of cytosolic proteases contributes to the processing. To test the importance of key lysine residues for mitochondrial targeting in mammalian cells, we transfected HEK293T cells with NTS DI2 2A, NTS DI2 5A, NTS DI2 7A, NTS DI2?K38A 40A and NTS DI2 29A 61A constructs. Again, we observed similar results compared to the situation in D. discoideum. NTS DI2 2A and NTS DI2 38A 40A are targeted to mitochondria, while NTS DI2 5A, NTS DI2 7A and NTS DI2 29A 61A are not targeted (Fig. S5). The non-targeted NTS DI2 5A, NTS DI2 7A and NTS DI2 29A 61A constructs display the same HDAC-IN-3 electrophoretic mobility as EGFP. This is most likely the result of nonspecific proteolytic degradation by cytosolic proteases that cleave off the exposed unfolded preprotein region from the tightly 
folded EGFP core (Fig. S6). In summary, our data show that the dynamin B presequence serves as an efficient targeting sequence in the DYm-dependent translocation of proteins from the cytosol into the mitochondrial matrix. The asparagine-rich region in the central part of the dynamin B presequence separates import sequences from processing sequences and does not seem to play a role in mitochondrial localization. Our results 1326631 define a minimal sequence formed by residues 28 to 64 that, in combination with mitochondrial protease cleavage sites, is sufficient for efficient protein targeting to mitochondria and proteolytic processing. The presence of a cluster of lysine residues on one side of the amphipathic helix is a key feature of the mitochondrial targeting sequence. Four properly positioned lysine residues are sufficient for effective targeting of a minimal construct. We demonstrate that the underlying mechanism of protein translocation from the cytoplasm into the mitochondrial matrix is evolutionarily conserved from social amoebae to humans.Supporting InformationFigure S1 Mitochondrial localization of dynamin B presequence deletion constructs. (A) Cells transformed with NTS DN1, (B) NTS DC and (C) NTS DI1 are shown. Cells were co-stained with mitoporin. Scale bars, 5 mm.Dictyostelium Mitochondrial Targeting Sequence(TIF)Figure S2 Mitochondrial localization of dynamin B presequence mutant constructs. (A) Cells transformed with NTS DI2, (B) NTS DI2 K2A, (C) NTS DI2 K38A-K40A, (D) NTS.Rix is mediated by a DYm-dependent mechanism (Fig. 6D). The residual mitochondrial signal can be explained by mitochondrial import prior to the CCCP treatment. Results describing the localization and processing of constructs carrying an altered version of the NTS in HEK293T cells are similar to those obtained for D. discoideum. Constructs NTS DRS and NTS 105A are targeted to mitochondria and proteolytically processed (Fig. S3). Multiple bands were observed on immunoblots when lysates from HEK293T cells expressing these proteins were analysed (Fig. S3E). This might be due to the combined 25033180 effects of MPP and MIP or differences in specificity compared to their D. discoideum counterparts. The minimal construct (NTS DI2) is properly processed and targeted to mitochondria in HEK293T cells. Processing of DI2 in HEK 293T cells appears to be more efficient than in D. discoideum and gives similar products. Deletion of the R-recognition site or introduction of mutation R105A reduces the targeting efficiency of NTS DI2 (Fig. S4A ). Compared to the situation in D. discoideum, NTS DI2 DRS and NTS DI2 R105A are more completely processed in HEK 293T cells (Fig. S4D). However, we cannot exclude that the unspecific action of cytosolic proteases contributes to the processing. To test the importance of key lysine residues for mitochondrial targeting in mammalian cells, we transfected HEK293T cells with NTS DI2 2A, NTS DI2 5A, NTS DI2 7A, NTS DI2?K38A 40A and NTS DI2 29A 61A constructs. Again, we observed similar results compared to the situation in D. discoideum. NTS DI2 2A and NTS DI2 38A 40A are targeted to mitochondria, while NTS DI2 5A, NTS DI2 7A and NTS DI2 29A 61A are not targeted (Fig. S5). The non-targeted NTS DI2 5A, NTS DI2 7A and NTS DI2 29A 61A constructs display the same electrophoretic mobility as EGFP. This is most likely the result of nonspecific proteolytic degradation by cytosolic proteases that cleave off the exposed unfolded preprotein region from the tightly folded EGFP core (Fig. S6). In summary, our data show that the dynamin B presequence serves as an efficient targeting sequence in the DYm-dependent translocation of proteins from the cytosol into the mitochondrial matrix. The asparagine-rich region in the central part of the dynamin B presequence separates import sequences from processing sequences and does not seem to play a role in mitochondrial localization. Our results 1326631 define a minimal sequence formed by residues 28 to 64 that, in combination with mitochondrial protease cleavage sites, is sufficient for efficient protein targeting to mitochondria and proteolytic processing. The presence of a cluster of lysine residues on one side of the amphipathic helix is a key feature of the mitochondrial targeting sequence. Four properly positioned lysine residues are sufficient for effective targeting of a minimal construct. We demonstrate that the underlying mechanism of protein translocation from the cytoplasm into the mitochondrial matrix is evolutionarily conserved from social amoebae to humans.Supporting InformationFigure S1 Mitochondrial localization of dynamin B presequence deletion constructs. (A) Cells transformed with NTS DN1, (B) NTS DC and (C) NTS DI1 are shown. Cells were co-stained with mitoporin. Scale bars, 5 mm.Dictyostelium Mitochondrial Targeting Sequence(TIF)Figure S2 Mitochondrial localization of dynamin B presequence mutant constructs. (A) Cells transformed with NTS DI2, (B) NTS DI2 K2A, (C) NTS DI2 K38A-K40A, (D) NTS.
