Multispan mitochondrial outer membrane protein Ugo1 follows a unique Mim1-dependent import pathway

Abstract

The mitochondrial outer membrane (MOM) harbors several multispan proteins that execute various functions. Despite their importance, the mechanisms by which these proteins are recognized and inserted into the outer membrane remain largely unclear. In this paper, we address this issue using yeast mitochondria and the multispan protein Ugo1. Using a specific insertion assay and analysis by native gel electrophoresis, we show that the import receptor Tom70, but not its partner Tom20, is involved in the initial recognition of the Ugo1 precursor. Surprisingly, the import pore formed by the translocase of the outer membrane complex appears not to be required for the insertion process. Conversely, the multifunctional outer membrane protein mitochondrial import 1 (Mim1) plays a central role in mediating the insertion of Ugo1. Collectively, these results suggest that Ugo1 is inserted into the MOM by a novel pathway in which Tom70 and Mim1 contribute to the efficiency and selectivity of the process.

Figure 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 protected from trypsin activity. The scissors represent the protease trypsin. (B) A proteolytic fragment of 23 kD is formed upon the correct insertion of Ugo1-2HA. Radiolabeled Ugo1-2HA was incubated for the indicated time periods with mitochondria isolated from ugo1Δ cells expressing plasmid-encoded Ugo1-2HA. Mitochondria were further incubated without (lane 2) or with trypsin (lanes 3–6) and pelleted. An additional sample was analyzed after import and trypsinization by carbonate extraction (Carb.), and pellet (P) and supernatant (S) fractions were loaded (lanes 7 and 8). In one sample, trypsin treatment was performed only after the mitochondria were solubilized with Triton X-100 (lane 9). As a control, 100% of input radiolabeled protein was treated with trypsin in the absence of mitochondria (lane 10). The proteolytic fragment (F) is indicated by black arrows, and full-length Ugo1 is indicated by white arrows. A nonspecific band resulting from preexisting mRNA in the reticulocyte lysate is indicated by the asterisks. In lane 11, mitochondria from the ugo1Δ strain harboring the empty plasmid were loaded as a control for the specificity of the HA antibody. All samples were analyzed by SDS-PAGE followed by autoradiography (top) and then immunodecorated with HA antibody (bottom). (C) The transcription–translation-coupled system was incubated with or without a plasmid encoding Ugo1-HA. In one sample, a commercial protease inhibitor cocktail was added. Samples were analyzed by SDS-PAGE and autoradiography. Full-length Ugo1 is indicated by a white arrow.

Figure 2.

Import of Ugo1 is strongly compromised by removal of ATP. (A and B) Radiolabeled Ugo1 (A) or pSu9-DHFR (B) was incubated in import buffer with mitochondria for the indicated time periods in the presence or absence of apyrase. As a control, samples without mitochondria (− Mitoch.) were incubated in the presence or absence of apyrase and analyzed directly by SDS-PAGE. At the end of the import reactions, mitochondria were treated with either trypsin (Ugo1 import reactions) or proteinase K (PK; pSu9-DHFR import reactions) and reisolated. Imported proteins were analyzed by SDS-PAGE and autoradiography. The insertion of Ugo1 was quantified by analyzing the formation of the 23-kD fragment (indicated by an arrow), whereas for pSu9-DHFR, the protease-protected mature form (m) was quantified. The amount of proteins imported into untreated mitochondria for the longest time period was set to 100%. An experiment representative of three independent repeats is presented. p, precursor.

Figure 3.

Ugo1 requires the import receptor Tom70 for its import and assembly. (A) Mitochondria were left intact or pretreated with trypsin followed by reisolation of the organelles. Aliquots of both trypsin-treated and intact mitochondria were removed, and the trypsin activity was monitored by immunodecoration with antibodies against Tom components (right). Next, radiolabeled Ugo1 and porin were incubated with the trypsin-treated or intact mitochondria for the indicated time periods. At the end of the import reactions, samples containing Ugo1 were treated again with trypsin, whereas to those harboring porin, PK was added. Proteins were analyzed by SDS-PAGE and autoradiography. The insertion of Ugo1 was quantified by analyzing the formation of the 23-kD fragment, whereas for porin, the PK-protected molecules were quantified. The amount of precursor proteins imported into intact mitochondria for 20 min was set to 100%. (B) Radiolabeled precursors were imported into mitochondria isolated from either wild-type (wt) or tom20Δ strains. Imported proteins were analyzed as described in A. (C) Radiolabeled precursors of Ugo1 and AAC were imported into mitochondria isolated from either wild-type or tom70Δtom71Δ strains. Imported proteins were analyzed by SDS-PAGE and radiography. The insertion of Ugo1 was quantified as described in A, whereas the PK-protected molecules of AAC were quantified. (A–C) An experiment representative of three independent repeats is presented. (D) Radiolabeled precursor of Ugo1 was imported into mitochondria isolated from tom70Δtom71Δ, tom20Δ, or their corresponding wild-type strains. After import, the mitochondria were analyzed by BN-PAGE. For comparison, wild-type mitochondria were analyzed by BN-PAGE and immunodecoration with an antibody against Ugo1. Ugo1-containing complexes are indicated on the right (I and II). (E) Mitochondria were isolated from tom70Δtom71Δ, tom20Δ, or their corresponding wild-type strains. Mitochondrial proteins (10 and 30 µg) were analyzed by SDS-PAGE and immunodecoration with antibodies against the indicated proteins. The intensity of the bands in three independent experiments was quantified, and the amount of proteins in mutant mitochondria is expressed as the mean (±SD) percentage of their level in the wild-type organelle. (F) The cytosolic domain of Tom70 can recognize newly synthesized Ugo1 molecules. Chemical amounts of Ugo1-HA (input) were mixed with either GST or GST fused to the cytosolic domain of Tom70 (GST-Tom70) bound to glutathione beads. The beads were washed, after which bound material was eluted with sample buffer. Aliquots of the input (10%), wash (W; 2%), and bound material (B; 100%) were analyzed by SDS-PAGE and either Ponceau staining (GST and GST-Tom70) or immunodecoration against HA tag.

Figure 4.

Mim1 is playing an important role in the membrane integration of Ugo1. (A) Radiolabeled Ugo1 and Tom40 were imported into mitochondria isolated from either wild-type (wt) or mim1Δ strains. The insertion of Ugo1 was analyzed as described in Fig. 3 A, whereas for Tom40, the PK-protected molecules were quantified. An experiment representative of three independent repeats is presented. (B) Radiolabeled precursor of Ugo1 was imported for the indicated time periods into mitochondria isolated from either wild-type or mim1Δ strains. After import, mitochondria were analyzed by BN-PAGE and autoradiography. Ugo1-containing complexes are indicated (I and II). (C) Mitochondria isolated from either wild-type or mim1Δ strains were analyzed by SDS-PAGE and immunodecoration with antibodies against the indicated mitochondrial proteins. The intensities of the bands were quantified as described in Fig. 3 E. (D) The indicated amounts of mitochondria isolated from either wild-type or mim1Δ strains were analyzed by BN-PAGE and immunodecoration with antibody against Ugo1. Ugo1-containing complexes are indicated. (E) Chemical amounts of Ugo1-HA (input) were mixed with either MBP or MBP-Mim1 bound to maltose beads. The beads were washed, and then bound material was eluted with sample buffer. Aliquots of the input (10%), wash (W; 2%), and bound material (B; 80%) were analyzed by SDS-PAGE and immunodecoration with antibodies against HA tag and MBP. (F) Mitochondria isolated from either wild-type or mim1Δ strains were subjected to carbonate extraction. The pellet (P) and the supernatant (S) fractions were analyzed by SDS-PAGE and immunodecoration with antibodies against the indicated proteins.