Tatsuta, T., and T. randomly chosen animals is usually shown. It is conceivable that this relative large quantity of Afg3l1 and Afg3l2 is usually altered in the absence of paraplegin, leading to a Mouse monoclonal antibody to ACE. This gene encodes an enzyme involved in catalyzing the conversion of angiotensin I into aphysiologically active peptide angiotensin II. Angiotensin II is a potent vasopressor andaldosterone-stimulating peptide that controls blood pressure and fluid-electrolyte balance. Thisenzyme plays a key role in the renin-angiotensin system. Many studies have associated thepresence or absence of a 287 bp Alu repeat element in this gene with the levels of circulatingenzyme or cardiovascular pathophysiologies. Two most abundant alternatively spliced variantsof this gene encode two isozymes-the somatic form and the testicular form that are equallyactive. Multiple additional alternatively spliced variants have been identified but their full lengthnature has not been determined.200471 ACE(N-terminus) Mouse mAbTel+ partial compensation for the loss of paraplegin. We therefore compared the relative large quantity of Afg3l1 and Afg3l2 in mitochondria isolated from liver and brain of em Spg7 /em ?/? mice lacking paraplegin (Fig. ?(Fig.4B).4B). When normalized to Afg3l1, Afg3l2 still accumulated at 10-fold-higher levels in brain mitochondria of em Spg7 /em ?/? mice, demonstrating that this relative large quantity of both proteins was not altered in the absence of paraplegin. These experiments reveal unexpected differences Artefenomel in Artefenomel the relative large quantity of em m /em -AAA protease subunits in different tissues. Considering the observed variability in their assembly, it appears likely that this subunit composition of the proteolytic complexes varies in a tissue-specific manner. Homo- and hetero-oligomeric em m /em -AAA proteases in brain mitochondria. To assess the assembly of em m /em -AAA protease subunits in a more quantitative manner, we performed immunodepletion experiments. Brain mitochondria were solubilized in digitonin, and affinity-purified Afg3l2-specific antibodies were used to completely deplete the extract of Afg3l2 (Fig. ?(Fig.5A).5A). Afg3l2 was immunologically not detectable in the supernatant portion after immunodepletion. Concomitant with Afg3l2, Afg3l1 and paraplegin were present at only drastically diminished levels (Fig. ?(Fig.5A).5A). The incubation of mitochondrial extracts with preimmune serum affected the steady-state level of neither Afg3l2 nor Afg3l1 or paraplegin (Fig. ?(Fig.5A).5A). We therefore conclude that Afg3l1, Afg3l2, and paraplegin assemble quantitatively with each other in the inner membrane of brain mitochondria. Open in a separate windows FIG. 5. Coexistence of em m /em -AAA proteases built up of different subunits in brain mitochondria. Mitochondria (150 g mitochondrial protein) from (A) wild-type (WT) and (B) em Spg7 /em ?/? brain were lysed in digitonin and incubated with saturating amounts of preimmune serum or affinity-purified Afg3l2- or Afg3l1C-specific antibodies. After the removal of the precipitate, supernatant fractions (SN) were analyzed by SDS-PAGE and examined for the presence of paraplegin, Afg3l1, and Afg3l2 by immunoblotting. A monoclonal antibody directed against the 39-kDa subunit of complex I (Ndufa9) was used to control for equivalent gel loading. Whereas Afg3l1 and Afg3l2 could be completely depleted from your supernatant fraction by using affinity-purified Afg3l1- or Afg3l2-specific antibodies, respectively, extracts could not be depleted of paraplegin using available paraplegin-specific antibodies (data not shown). Notably, when comparable experiments were performed with Afg3l1-specific antibodies, Afg3l2 and paraplegin were only slightly depleted from mitochondrial extracts. This most likely displays the decreased large quantity of Afg3l1 compared to that of Afg3l2 and paraplegin. Thus, at least two different em m /em -AAA protease complexes coexist in brain mitochondria: Afg3l2/paraplegin and less abundant Afg3l1 complexes that also contain Afg3l2, paraplegin, or both. Which em m /em -AAA protease complexes are present in paraplegin-deficient brain mitochondria? To address this question, we isolated brain mitochondria from em Spg7 /em ?/? mice and analyzed the complex composition of em m /em -AAA protease complexes by immunodepletion using Afg3l1- and Afg3l2-specific antibodies (Fig. ?(Fig.5B).5B). Much like wild-type mitochondria, Afg3l1 was almost completely depleted from extracts of em Spg7 /em ?/? brain mitochondria with Afg3l2 antibodies demonstrating quantitative assembly (Fig. ?(Fig.5B).5B). In contrast, the steady-state level of Afg3l2 was hardly affected by depleting Afg3l1 (Fig. ?(Fig.5B).5B). We conclude that Afg3l2 is present in excess relative to Afg3l1 in brain mitochondria of wild-type and em Spg7 /em ?/? mice. While Afg3l1 is usually quantitatively put together with Afg3l2, the majority of Afg3l2 is not part of this structure and most likely forms a homo-oligomeric complex. Proteolytic activity of homo- and hetero-oligomeric em m /em -AAA proteases. These experiments demonstrate the presence of em m /em -AAA protease complexes with variable subunit composition in murine mitochondria. To examine the proteolytic activity of the different assemblies, we carried out complementation studies with em m /em -AAA protease-deficient em yta10 /em em yta12 /em yeast cells. In initial experiments, paraplegin, Afg3l1, and Afg3l2 were independently expressed in em yta10 /em em yta12 /em cells and respiratory cell growth was examined (Fig. ?(Fig.6A).6A). Strikingly, the expression Artefenomel of either Afg3l1 or Afg3l2 but not paraplegin restored the growth of em yta10 /em em yta12 /em cells on glycerol-containing medium (Fig. ?(Fig.6A).6A). This is reminiscent of hAFG3L2, which created homo-oligomeric, proteolytically active complexes when expressed in yeast (Fig. ?(Fig.1).1). Gel filtration analysis of mitochondrial extracts indeed revealed homo-oligomerization of Afg3l1 and Afg3l2 in yeast, whereas paraplegin did not form a high-molecular-mass complex in the absence of other AAA protease subunits (data not shown). The expression of proteolytic site variants of Afg3l1 (Afg3l1E567Q) or Afg3l2 (Afg3l2E574Q) did not promote respiratory growth of em yta10 /em em yta12 /em cells, indicating that the homo-oligomeric complexes exert proteolytic activity (Fig. ?(Fig.6A).6A). Consistently, we observed the maturation of MrpL32 in em yta10 /em em yta12 /em cells harboring Afg3l1 or Afg3l2, whereas this activity was abolished by mutations in the proteolytic center of each protein (Fig. ?(Fig.6B).6B). Moreover,.
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