In addition, our results show that the turnover of TbYme1-Myc in TbSlp2?/? parasites (t1/2 6

In addition, our results show that the turnover of TbYme1-Myc in TbSlp2?/? parasites (t1/2 6.3?h) was significantly decreased compared to wild-type Vinpocetine cells (t1/2 1.8?h) (Figure 4D). Open in a separate window FIGURE 4 TbSlp2 and TbYme1 are interdependent. for growth of parasites. In addition, we demonstrate that TbSlp2 binds to the metalloprotease TbYme1 and together they form a large mitochondrial protein complex. The two proteins negatively regulate each others expression levels by accelerating protein turnover. Furthermore, we show that TbYme1 plays a role in heat-stress resistance, as TbYme1 knock-out parasites displayed mitochondrial fragmentation and loss of viability when cultured at elevated temperatures. Unbiased interaction studies uncovered putative TbYme1 substrates, some of which were differentially affected by the absence of TbYme1. Our results support emerging evidence for the presence of mitochondrial quality control pathways in this ancient eukaryote. is a unicellular protozoan parasite causing human African Trypanosomiasis, also known as sleeping sickness, and nagana in domestic animals in Sub-Saharan Africa. is an established model organism to study eukaryotic cell biology (Btikofer et al., 2001; Fairlamb et al., 2016; Schneider and Ochsenreiter, 2018) and lipid Vinpocetine metabolism (Serricchio and Btikofer, 2010; Ramakrishnan et al., 2013). This highly diverged eukaryote is unrelated to Opisthokonts (Walker et al., 2011) and thus provides a unique opportunity to study ancestral functions of organelles and proteins (Btikofer et al., 2001; Schneider, 2018; Schneider and Ochsenreiter, 2018) that are conserved in higher eukaryotes. Two enzymes of the CL biosynthetic pathway have been identified and studied in phosphatidylglycerophosphate synthase (TbPgs) Cav1.2 and cardiolipin synthase (TbCls) are essential for CL biosynthesis, mitochondrial function and parasite survival (Serricchio and Btikofer, 2012; 2013). Here, we identify and characterize the SLP-2 homolog (TbSlp2) and show that, in contrast to human, it is dispensable for mitochondrial health. TbSlp2 localizes to mitochondria where it binds to membranes via phosphatidic acid (PA) and interacts with prohibitin 1 (TbPhb1) and TbPgs, possibly linking CL biosynthesis to CL microdomain formation. Moreover, we demonstrate that TbSlp2 forms a protozoan SPY-like complex with a newly identified YME1L homolog (TbYme1). Interestingly, TbSlp2 and TbYme1 negatively regulate each other and are involved in mitochondrial stress response by acting as pro-survival proteins. Results Stomatin-like Protein 2 is Conserved in genome encodes three proteins containing SPFH domains, TbPhb1, TbPhb2 and a putative stomatin-like protein (Tb927.5.520). Blast searches with the putative stomatin-like protein against the human proteome revealed the most significant alignment with SLP-2/STOML2. Pairwise sequence alignment of the deduced full-length stomatin-like protein (TbSlp2) with human being SLP-2 exposed an overall sequence identity of 30% (41% sequence similarity). The stomatin website alone exposed a 57% sequence identity and a 74% sequence similarity with human being SLP-2. The deduced TbSlp2 protein has a determined molecular mass of 56?kDa, contains a conserved SPFH website and a C-terminal website, but lacks transmembrane domains or membrane hairpins (Lapatsina et al., 2012) (Number 1A). Open in a separate window Number 1 Characterization of TbSlp2 in procyclic forms. (C) procyclic forms expressing TbSlp2-HA were incubated with antibodies against HA and the mitochondrial ADP/ATP carrier (TbAAC) and analyzed by fluorescence microscopy. Level pub: 5?m (D) Carbonate extraction of TbSlp2-HA. Mitochondrial Vinpocetine membranes were treated with 0.1?M Na2CO3 at pH 10.5, 11.5 and 12.5, separated by ultracentrifugation into soluble (S) and membrane (P) fractions, and analyzed by immunoblotting using primary antibodies against HA, TbHsp60 (soluble matrix protein), or TbPhb1 and TbAAC (integral membrane proteins). (E) TbSlp2-HA isolated from procyclic forms expressing C-terminally HA-tagged TbSlp2 using their genomic locus (henceforth called tagged TbSlp2-HA) and analyzed total parasite protein by SDS-PAGE and immunoblotting. The results display that TbSlp2-HA migrated as a single 55?kDa band (Number 1B). Subsequently, the subcellular localization of TbSlp2-HA was analyzed using immunofluorescence microscopy. Co-staining of parasites expressing TbSlp2-HA using antibodies against HA and the mitochondrial ADP/ATP carrier (TbAAC) exposed that TbSlp2-HA localizes to the mitochondrion (Number 1C). Members of the SPFH.