1 B, lanes 7 and 8). put into the MOM by a novel pathway in which Tom70 and Mim1 contribute to the effectiveness and selectivity of the process. Intro The mitochondrial outer membrane (MOM) consists of a diverse set of proteins with numerous functions (Burri et al., 2006; Schmitt et al., 2006; Zahedi et al., 2006). All of these proteins, like the vast majority of mitochondrial proteins, are nuclear encoded, synthesized in Pyridostatin the cytosol, and imported into the organelle (Neupert and Herrmann, 2007; Chacinska et al., 2009; Endo and Yamano, 2009; Walther and Rapaport, 2009). Multispan proteins comprise a distinct class of MOM proteins and are integrated into the lipid bilayer via multiple transmembrane segments (TMSs). Some of them, like Fzo1 in candida (Mfn1/2 in mammals), mix the membrane twice, exposing N- and C-terminal domains FLJ20285 toward the cytosol (Fritz et al., 2001; Rojo et al., 2002). Additional multispan MOM proteins with three or more TMSs are Ugo1 and OM14 in candida and members of the Bcl-2 family and human being peripheral benzodiazepine receptor (PBR) in higher eukaryotes (Burri et al., 2006; Coonrod et al., 2007; Otera et al., 2007; Hoppins et al., 2009; Chipuk et al., 2010). Earlier study using mutants of Ugo1 and Mfn2 exposed that the website that includes the expected TMS harbors Pyridostatin the information necessary for mitochondrial focusing on, although additional focusing on signals in additional regions of the protein could not become excluded (Rojo et al., 2002; Coonrod et al., 2007). Experiments with Mfn2 show similarities between polytopic and tail-anchored (TA) proteins in terms of motifs and mechanisms responsible for their insertion into MOM (Rojo et al., 2002). The idea that import pathways of TA and multispan proteins overlap (at least partially) is supported by import competition assays in which import of PBR was strongly inhibited by an excess amount of the TA protein Bak (Otera et al., 2007). However, in contrast to the biogenesis of TA proteins in which import receptors are probably not essential for the process (Setoguchi et al., 2006; Kemper et al., 2008), such receptors appear to play a role in the membrane integration of multispan proteins. Fzo1 requires protease-sensitive import receptors for its insertion into the MOM (Rapaport et al., 1998), and later on investigations exposed that import of PBR and Mfn2 requires connection with Tom70 but is definitely independent of additional translocase of the outer membrane (TOM) parts (Otera et al., 2007; Yamano et al., 2008). Despite the aforementioned progress, the mechanisms by which newly synthesized multispan proteins are recognized in the organelle surface and inserted into the MOM are still mainly unresolved. It is especially unclear whether dedicated membrane insertion machinery for such proteins is Pyridostatin present. To address these questions, we analyzed the Pyridostatin membrane integration of the model multispan protein Ugo1. Our results suggest a novel insertion pathway in which the mitochondrial import receptor Tom70 and the outer membrane protein mitochondrial import 1 (Mim1) regulate acknowledgement and insertion of Ugo1. Results and discussion A specific assay to monitor the in vitro insertion of Ugo1 A long-lasting problem in analyzing the integration of multispan proteins is the lack of a reliable assay Pyridostatin for right membrane integration. To overcome this problem, we developed a proteolytic assay based on earlier experiments suggesting Ugo1 to have at least three TMSs (Coonrod et al., 2007; Hoppins et al., 2009). These earlier observations and our current results indicate the addition of trypsin to mitochondria comprising C-terminally HA-tagged Ugo1 resulted in the formation of a 23-kD C-terminal fragment probably as a result of a proteolytic cleavage site between putative TMS 2 and 3 (Fig. 1 A). Of notice, C-terminally HA-tagged Ugo1 is definitely fully functional and thus has a nativelike topology (Hoppins et al., 2009). Open in a separate window Number 1. A novel assay to study in vitro import of Ugo1. (A) A schematic representation of 2HA-tagged Ugo1 (Ugo1-2HA) and Ugo1-2HA C-terminal 23-kD fragment safeguarded from trypsin activity. The scissors represent the protease trypsin. (B) A proteolytic fragment of 23 kD is definitely.
Decarboxylases