Volume 56, Issue 5, Pages 641-652 (December 2014) Mgr2 Functions as Lateral Gatekeeper for Preprotein Sorting in the Mitochondrial Inner Membrane  Raffaele Ieva, Sandra G. Schrempp, Łukasz Opaliński, Florian Wollweber, Philipp Höß, Anna K. Heißwolf, Michael Gebert, Ying Zhang, Bernard Guiard, Sabine Rospert, Thomas Becker, Agnieszka Chacinska, Nikolaus Pfanner, Martin van der Laan  Molecular Cell  Volume 56, Issue 5, Pages 641-652 (December 2014) DOI: 10.1016/j.molcel.2014.10.010 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 56, 641-652DOI: (10.1016/j.molcel.2014.10.010) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 Mgr2 Is Part of the Active Preprotein Translocase (A and B) 35S-labeled Tim23, Tim21, or Mgr2 was imported into mitochondria prior to or after accumulation of methotrexate (MTX)-bound, purified b2(167)Δ-DHFR in TOM-TIM23 supercomplexes (A, schematic presentation). Samples were analyzed by blue native PAGE (B, lanes 1–16, assembly of 35S-labeled proteins into the supercomplex) or treated with proteinase K and analyzed by SDS-PAGE (B, lanes 17–28, import of 35S-labeled proteins into mitochondria), followed by digital autoradiography. Nonassembled precursors migrated outside of the separation range of the blue native gels shown here. b2Δ-DHFR, b2(167)Δ-DHFR; IM, inner membrane; IMS, intermembrane space; OM, outer membrane. (C) Quantification of radiolabeled TOM-TIM23 supercomplexes detected by blue native PAGE as in (B) upon import of [35S]Tim23, [35S]Tim21, or [35S]Mgr2 before (first import, set to 100%) or after (second import) accumulation of MTX-bound b2(167)Δ-DHFR. Data are represented as mean ± SEM (n = 3). See also Figure S1. Molecular Cell 2014 56, 641-652DOI: (10.1016/j.molcel.2014.10.010) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 Mgr2 Is in Close Proximity to Translocation-Arrested Preproteins (A and B) 35S-labeled b2(110)Δ-DHFR (A) or b2(84)-DHFR (B) with a photoactivatable Nε−(5-azido-2-nitrobenzoyl)-lysine residue (ANB-lysine) at the indicated positions (pos) was accumulated in mitochondrial import sites in the presence of methotrexate and UV-irradiated. Samples were subjected to immunoprecipitation (IP) with Mgr2-specific antibodies. Elution fractions were analyzed by SDS-PAGE and autoradiography. Mgr2∗, Mgr2-containing crosslinking products; p, precursor; i, intermediate. (C) Quantitative analysis of Mgr2-containing site-specific crosslinking products (IP) shown in (A) and (B). Values were normalized for the amount of imported and processed substrate in each reaction and plotted against the position of crosslinker incorporation. The highest crosslinking yield obtained for position 47 of b2(84)-DHFR was set to 10. The sequences of the amino acid positions 45–71 of the preprotein constructs are depicted in blue for b2(110)Δ-DHFR and in red for b2(84)-DHFR. b2(110)Δ-DHFR contains a 19-residue deletion, leading to a shift of the amino acid positions (see Figure S1A for amino acid numbers according to the cytochrome b2 sequence). Targ, targeted. See also Figure S2. Molecular Cell 2014 56, 641-652DOI: (10.1016/j.molcel.2014.10.010) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 Mgr2 Expression Levels Influence the Rate of Preprotein Sorting into the Inner Membrane (A and B) Analysis of the import-driving activity exerted by the TIM23/PAM machinery on matrix-targeted b2(167)Δ-DHFR and inner membrane-sorted b2(220)-DHFR in wild-type (WT), mgr2Δ (A), and ssc1-3 (B) mitochondria. The 35S-labeled preproteins were incubated with mitochondria (Mito) in the presence of MTX, followed by a second incubation (Δt) in the absence of Δψ and treatment with proteinase K (Prot. K) as indicated. m, mature. (C) Membrane sorting of b2(220)-DHFR was analyzed by an import/chase assay in wild-type, mgr2Δ, and Mgr2 overexpression (Mgr2↑) mitochondria. The preprotein was accumulated in the presence of dihydrofolate (DHF) and NADPH, followed by reisolation of mitochondria, a second incubation (chase), and treatment with proteinase K. Formation of the mature form during chase periods (Δt) was monitored by SDS-PAGE and autoradiography. Processed, i + m forms. Data are represented as mean ± SEM (n = 3). See also Figure S3. Molecular Cell 2014 56, 641-652DOI: (10.1016/j.molcel.2014.10.010) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Mgr2 Stabilizes Preprotein Accumulation in the TIM23 Complex (A) Radiolabeled b2(167)Δ-DHFR or b2(220)-DHFR was accumulated in import sites of isolated mitochondria in the presence of MTX, and samples were analyzed by blue native or SDS-PAGE and autoradiography. (B and C) Preprotein-loaded mitochondria generated as in (A) were subjected to osmotic swelling and incubated with the indicated antibodies prior to blue native PAGE analysis. (D) b2(220)-DHFR was incubated with mitochondria in the presence or absence of Δψ and MTX (lanes 1–4). Shown is the time course of b2(220)-DHFR accumulation in WT, tim17-4, or tim17-5 mitochondria in the presence of Δψ and MTX (lanes 5–19). (E) The import-driving activity generated on b2(220)-DHFR was analyzed in WT and Mgr2 overexpression (Mgr2↑) mitochondria as in Figure 3A. The proteinase K-resistant intermediate was quantified by digital autoradiography. Data are represented as mean ± SEM (n = 3). (F) Accumulation of b2(220)-DHFR in import sites of the indicated mitochondria as in (A). See also Figure S4. Molecular Cell 2014 56, 641-652DOI: (10.1016/j.molcel.2014.10.010) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 Mgr2 Promotes Quality Control of Inner Membrane Sorting (A) [35S]b2(220)IC-DHFR was imported into WT, mgr2Δ, or tim21Δ mitochondria and treated with proteinase K where indicated. The samples were analyzed by SDS-PAGE and autoradiography. (B and C) Accumulation of radiolabeled b2(220)IC-DHFR in import sites of WT or mgr2Δ (B) or WT and Mgr2K18A,K20A (C) mitochondria as described in Figure 4A. See also Figure S5. Molecular Cell 2014 56, 641-652DOI: (10.1016/j.molcel.2014.10.010) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 Mgr2 Regulates Inner Membrane Sorting and Alternative Topogenesis of Mgm1 (A) WT and mgr2Δ mitochondria were analyzed by SDS-PAGE and immunoblotting using the indicated antisera. The ratio of long to short Mgm1 forms was quantified digitally. Data are represented as mean ± SEM (n = 3). Asterisk, nonspecific band. (B) Morphology of WT and mgr2Δ mitochondria visualized by 3,3′-dihexyloxacarbocyanine iodide staining and fluorescence microscopy. Scale bars, 5 μm. The percentage of cells with tubular mitochondrial network is shown as mean ± SEM (three independent cultures and ≥ 300 cells/strain). YPD, yeast extract-peptone-dextrose. (C) Quantitative analysis of mitochondrial morphology in WT cells compared with mgr2Δ cells with or without overexpression of Mgm1 performed as in (B). (D) Protein content of WT and Mgr2 overexpression mitochondria (Mgr2↑) analyzed as in (A). (E) Mitochondrial morphology in WT and Mgr2-overexpressing (Mgr2↑) cells analyzed and quantified as in (B). Scale bars, 5 μm. Molecular Cell 2014 56, 641-652DOI: (10.1016/j.molcel.2014.10.010) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 Mgr2 Acts as a Lateral Gatekeeper of the TIM23 Complex Hypothetical model illustrating the gatekeeping function of Mgr2 in the TIM23 complex. Mgr2 may constitute a physical part of the gate. Molecular Cell 2014 56, 641-652DOI: (10.1016/j.molcel.2014.10.010) Copyright © 2014 Elsevier Inc. Terms and Conditions