Targeting of Phb2 to Mitochondria by a Bipartite Noncleavable Presequence
Although generally highly conserved from yeast to human, both Phb1 and Phb2 are more divergent in their N-terminal regions. For Phb2, this segment is composed of a hydrophilic stretch followed by a putative hydrophobic transmembrane domain. Positively charged amino acid residues are enriched within the first 35 amino acid residues, which can form an amphipatic α-helix, a characteristic of mitochondrial sorting sequences (). To examine a potential targeting function of this segment, a series of Phb2 variants were constructed (), synthesized in a cell-free system in the presence of [35S]methionine, and analyzed for their capability to be imported into isolated mitochondria in vitro. Phb2 became resistant to externally added protease in a membrane-potential dependent manner without proteolytic removal of a targeting sequence (). Mitochondrial import, however, was impaired upon deletion of the N-terminal 61 amino acid residues of Phb2 [Phb2(62-310)] demonstrating that this segment harbors essential targeting information (). To dissect the role of hydrophilic and hydrophobic stretches in this segment, we analyzed mitochondrial import of Phb2(36-310) and Phb2(Δ36-61), lacking the positively charged or the putative transmembrane region, respectively. Import of Phb2(36-310) was impaired and occurred with significantly reduced kinetics when compared with Phb2 (). Thus, although the positively charged, N-terminal segment indeed ensures efficient mitochondrial import, additional targeting information is provided by the subsequent hydrophobic segment. Consistently, deletion of the corresponding amino acid residues 36-61 in Phb2 also impaired mitochondrial import (). Notably, Phb2(Δ36-61) did not accumulate within mitochondria, indicating rapid degradation of the newly imported protein.
Figure 1. Targeting of Phb2 to mitochondria by a noncleavable, bipartite presequence at the N terminus. (A) N-terminal region of Phb2. Amino acid residues 1-70 of S. cerevisiae Phb2 and a helical wheel representation of amino acid residues 8-25 is shown. Charged (more ...)
To substantiate these findings, we constructed hybrid proteins composed of N-terminal segments of Phb2 and mouse dihydrofolate reductase (DHFR). Phb2(1-61)-DHFR was efficiently imported into mitochondria, demonstrating that the N-terminal 61-amino acid residues are sufficient for mitochondrial targeting (). In contrast, import of Phb2(1-35)-DHFR or Phb2(36-61)-DHFR occurred with low efficiency, indicating that neither the positively charged nor the hydrophobic segment is able to ensure efficient mitochondrial import of a heterologous protein ().
We conclude from these experiments that mitochondrial targeting of Phb2 is ensured by a bipartite noncleavable presequence at the N terminus of Phb2, which is composed of a positively charged and a hydrophobic protein segment.
Mitochondrial Targeting of Phb1 by an Unconventional N-Terminal Presequence
Similar to Phb2, Phb1 subunits expose large domains to the intermembrane space and are thought to be anchored N-terminally to the inner membrane. However, neither a positively charged amino acid stretch with a propensity to form a amphipathic α-helix nor a hydrophobic segment predicted to form a membrane-spanning domain are present within the N-terminal region of Phb1 (). Nevertheless, deletion of 28 N-terminal amino acid residues of Phb1 [Phb1(29-287)] abolished import into isolated mitochondria, indicating that the N-terminal segment is indispensable for mitochondrial targeting ().
Figure 2. Mitochondrial targeting of Phb1 by an unconventional N-terminal presequence. (A) N-terminal region of Phb1. Amino acid residues 1-40 of S. cerevisiae Phb1 and a helical wheel representation of amino acid residues 1-18 is shown and marked as in (more ...)
When fused to mouse DHFR, however, the 28 N-terminal amino acid residues of Phb1 were not sufficient to promote import of the hybrid protein (). Prolongation of the Phb1-moiety to 57 amino acid residues did not affect targeting to mitochondria, whereas we observed significant import of Phb1(1-83)-DHFR into mitochondria (). To examine a possible impairment of mitochondrial import by folding of the DHFR-moiety, the hybrid proteins also were incubated with isolated mitochondria after denaturation in urea-containing buffer. Under these conditions, Phb1(1-28)DHFR was efficiently imported demonstrating that the first 28 N-terminal amino acid residues contain the complete targeting information (). These experiments demonstrate that Phb1 subunits are targeted to mitochondria by an unconventional noncleavable targeting sequence present at their N terminus.
Membrane Insertion Is Mediated by the TIM23-Translocase
The inner membrane harbors two protein translocases for nuclear-encoded preproteins: the TIM23-translocase for presequence-carrying precursor proteins and the TIM22-translocase for polytopic inner membrane proteins (for reviews, see Neupert, 1997
; Pfanner and Geissler, 2001
). To examine a potential role of the TIM23-translocase for the biogenesis of prohibitins, we used a yeast strain expressing Tim23, a major component of this translocase, from a galactose-inducible promoter. Cells were grown on galactose-free medium for Tim23 depletion and the accumulation of mitochondrial proteins was assessed by immunoblotting. Phb1 and Phb2 were hardly detectable in mitochondria depleted of Tim23, indicating that both proteins require the TIM23-translocase for membrane insertion and stable accumulation within mitochondria (). Similarly, presequence-containing matrix proteins, like the α-subunit of the F1
-ATPase, were not detectable in these cells (). In contrast, the carrier protein Aac2, which is imported by the TIM22-translocase (Sirrenberg et al., 1996
), accumulated at normal levels in Tim23-depleted mitochondria, demonstrating that the TIM22 pathway was not affected under these conditions ().
Figure 3. Requirement of the TIM23-translocase for prohibitin biogenesis. (A) Depletion of Tim23 in vivo. Mitochondial membranes were prepared from Tim23(Gal10) cells that were shifted for 24 h to galactose-free medium (Tim23↓). Samples were analyzed by (more ...)
Because Phb1 and Phb2 are functionally interdependent (Berger and Yaffe, 1998
; see below), an impaired membrane insertion of either subunit might indirectly affect the accumulation of the other subunit in Tim23-depleted mitochondria. We therefore analyzed the import of radiolabeled Phb1 and Phb2 into mitochondria expressing a C-terminally truncated, mutant variant of Tim23 [Tim23(fs)] (Sirrenberg et al., 1996
). Mitochondria isolated from these cells harbored decreased levels of Tim23, whereas Tim22 accumulated at normal levels (Sirrenberg et al., 1996
; data not shown). The import of both proteins into these mitochondria was strongly impaired when compared with wild-type mitochondria (). Control experiments demonstrated a general deficiency of TIM23-mediated import, whereas the TIM22-dependent pathway was still functional: the import of the matrix-localized preprotein Su9-DHFR into Tim23(fs) mitochondria was impaired, whereas Tim23, by also using the TIM22-translocase (Leuenberger et al., 1999
), accumulated with similar efficiencies in Tim23(fs) and wild-type mitochondria (). Depletion of Tim22 from mitochondria, on the other hand, did not affect import of Phb1 or Phb2 (our unpublished data). We conclude from these experiments that both Phb1 and Phb2 are inserted into the inner membrane by the TIM23-translocase.
Formation of an Assembly Intermediate Containing Phb1 and Phb2
We next analyzed complex formation of Phb1 and Phb2 by chemical cross-linking. 35S-labeled Phb1 was imported into mitochondria that were subsequently incubated with disuccinimidyl glutarate (DSG) (). Phb1-containing cross-link adducts of 39, 58, and 87 kDa accumulated in these mitochondria in a dose-dependent manner (). Several lines of evidence demonstrate an association of newly imported Phb1 with Phb2. First, Phb2-containing complexes of 58 and 87 kDa accumulated upon cross-linking in these mitochondria as revealed by immunoblot analysis by using Phb2-specific antisera (). Second, the formation of these cross-link adducts was found to depend on the presence of Phb2 but not of Phb1, indicating that Phb1-homo-oligomers are not formed. Similar-sized complexes were detected in mitochondria isolated from cells that strongly overexpress and therefore accumulate Phb2, despite the absence of Phb1 (). On the other hand, only the 39-kDa adduct was formed in Δphb1Δphb2 mitochondria (). Third, although with low efficiency, the 87- and 58-kDa cross-link adducts were precipitated with Phb2 antiserum but not with preimmune serum (). We therefore conclude that newly imported Phb1 assembles with Phb2. To examine the formation of large complexes by newly imported Phb1, mitochondria were solubilized after completion of import and chemical cross-linking and extracts were analyzed by two-dimensional blue native/SDS-polyacrylamide gel electrophoresis (BN/SDS-PAGE) (). Whereas preexisting prohibitin complexes were recovered as large protein assemblies (, middle), newly imported Phb1 formed a broad peak, indicating inefficient assembly (). The fractionation was not affected by the presence of excess level of Phb2 within mitochondria (our unpublished data). The cross-link adducts, however, were detected in two distinct complexes with native molecular masses of ~1-2 MDa and of ~120 kDa, respectively (). The larger form comigrated with the endogenous prohibitin complex and therefore most likely represents the fully assembled complex.
Figure 4. Intermediate-sized prohibitin complexes containing Phb1 and Tim13 or Phb1 and Phb2. (A) Chemical cross-linking of newly imported Phb1. After import of 35S-labeled Phb1 and trypsin digestion, mitochondria were subjected to chemical cross-linking by using (more ...)
Strikingly, the 39-kDa cross-link adduct was detected in the ~120-kDa intermediate-sized complex, indicating a transient interaction of a small mitochondrial protein with newly imported Phb1 (). Several low-molecular-weight Tim proteins have been linked to the import of nuclear-encoded mitochondrial proteins (Koehler, 2004
). Whereas the Tim9/10 complex is involved in the import of polytopic inner membrane proteins, the Tim8/13 complex plays a role for insertion of some proteins into both the inner or outer membrane (Davis et al., 2000
; Paschen et al., 2000
; Curran et al., 2002
; Vasiljev et al., 2004
). To examine a potential involvement of either complex in the biogenesis of prohibitins, coimmunoprecipitation experiments were performed after import of Phb1 into mitochondria and chemical cross-linking. Whereas no cross-link adducts were precipitated with Tim10-specific antiserum, the 39-kDa form was efficiently precipitated with Tim13-specific antiserum, indicating an interaction of Phb1 with the Tim8/13 complex upon import (). Notably, binding of Phb1 to Tim13 did not depend on the presence of Phb2 within mitochondria and therefore seems to occur before the formation of the Phb1/Phb2 assembly intermediate (, bottom).
The interaction of Phb1 with the Tim8/13 complex, however, is not essential for the biogenesis of the fully assembled prohibitin complex. Phb1 and Phb2 accumulated in Δtim8Δtim13 mitochondria at similar levels as in wild-type mitochondria (our unpublished data). Moreover, import of Phb1 occurred with similar efficiencies in wild-type and Δtim8Δtim13 mitochondria and was not affected by lowering the membrane potential across the inner membrane (our unpublished data).
The cross-linking experiments indicate that two ~120-kDa complexes are formed upon import of Phb1: a Phb1/Tim13 complex and a Phb1/Phb2 complex that may represent an assembly intermediate. To substantiate this conclusion, we analyzed the time course of its accumulation within mitochondria by BN/PAGE (). Radiolabeled Phb1 was imported into wild-type mitochondria that were further incubated after completion of import to allow a more efficient formation of the prohibitin complex. Assembly of newly imported Phb1 was assessed by BN/PAGE (). Whereas 120-kDa forms vanished over time, we observed the formation of the assembled prohibitin complex upon prolonged incubation of mitochondria (). As membranes were solubilized under mild conditions by using digitonin in these experiments, the supercomplex of prohibitins with the m-AAA protease was detected on the BN/PAGE (). The formation of Phb1-containing complexes depended on the membrane potential across the inner membrane and thus seems to require membrane insertion. The 120-kDa form was not detectable after import of Phb1 into Δphb1Δphb2 mitochondria, indicating that these forms represent mainly Phb1/Phb2 complexes (). Consistently, their formation was only slightly affected in mitochondria isolated from Δtim8Δtim13 cells (our unpublished data). These experiments demonstrate the transient accumulation of 120-kDa Phb1/Phb2 complexes upon import of Phb1 into mitochondria and suggest that this form represents an assembly intermediate.
Assembly of the Prohibitin Complex Depends on Coiled-Coil Regions in Phb1 and Phb2
The only conserved sequence motif detectable in prohibitin subunits is a predicted coiled-coil region in the C-terminal part of the polypeptides (amino acid residues 180-224 of Phb1 and 212-253 of Phb2) whose function is presently not understood. The C-terminally truncated variants Phb1(1-180) and Phb2(1-213) were efficiently imported into isolated mitochondria, demonstrating that C-terminal parts of both subunits are dispensable for mitochondrial targeting and transport (Figures , and ). Notably, although occurring with initial rates similar to Phb1, import of Phb1(1-180) ceased upon prolonged incubation times (). This might be explained by a decreased solubility of Phb1(1-180) before import or reflect proteolysis of newly imported Phb1(1-180) within mitochondria.
Phb1 and Phb2 are interdependent and accumulate stably only in an assembled state, e.g., nonassembled subunits are rapidly degraded within mitochondria (Berger and Yaffe, 1998
). We therefore examined a potential role of the C-terminal coiled-coil regions in both subunits for the assembly of the prohibitin complex. Termination codons were genomically inserted in the PHB1
genes to allow the expression of C-terminally truncated Phb1(1-180) or Phb2(1-191) lacking the coiled-coil regions. In agreement with an impaired assembly of the prohibitin complex, Phb1 or Phb2 did not accumulate in cells expressing Phb2(1-191) or Phb1(1-180) (). We also did not observe a stabilization of Phb1 upon modest overexpression of Phb2(1-216) in Δphb2
cells, nor of Phb2 in the presence of increased levels of Phb1(1-180) in Δphb1
cells (). These results point to an essential function of C-terminal coiled-coil regions in Phb1 and Phb2 for the assembly of the prohibitin complex.
Figure 5. Assembly of the prohibitin complex depends on C-terminal regions of Phb1 and Phb2. (A and B) Mitochondrial membranes were analyzed by SDS-PAGE and immunoblotting by using Phb1- and Phb2-specific, and as a loading control, Yme1-specific antisera in the (more ...)
Surprisingly, deletion of only four or three C-terminal amino acid residues from genomically encoded Phb1 or Phb2, respectively, caused already a destabilization of the respective binding partner (), indicating that the integrity of the C-terminal end of both prohibitin subunits is important for complex formation. Consistently, heaxahistidine peptides fused to the C terminus of Phb1 or Phb2 abolished the assembly of the prohibitin complex (our unpublished data). The impaired complex formation after deletion of short C-terminal segments of Phb1 or Phb2 could be overcome by modest overexpression of Phb2(1-303) or Phb1(1-283), demonstrating that, in contrast to the coiled-coil regions, C-terminal amino acid residues of Phb1 and Phb2 are crucial but not essential for the assembly of the prohibitin complex.
The Coiled-Coil Region of Phb1 Is Required for the Formation of Large Complexes but Dispensable for the Interaction with Phb2 and Tim13
The detection of Phb1/Phb2 assembly by chemical cross-linking and BN/PAGE analyses allowed us to further define the role of the coiled-coil region of Phb1 during the formation of the prohibitin complex. 35S-labeled Phb1 and Phb1(1-180) lacking the C-terminal coiled-coil domain were imported into isolated mitochondria. After completion of import and solubilization of mitochondrial membranes with digitonin, the assembly of the newly imported proteins was assessed by BN/SDS-PAGE (). As observed previously (), Phb1 was detected as a broad peak after electrophoretic fractionation of mitochondrial extracts. It should be noted, however, that small portions of newly imported Phb1 were enriched in intermediate-sized complexes and in a large complex, indicating assembly of the ring complex (). In contrast, assemblies containing Phb1(1-180) were not detected (). These findings are in agreement with the in vivo analysis and indicate that the coiled-coil region of Phb1 is essential for the formation of the prohibitin complex.
Chemical cross-linking was used to examine the requirement of the coiled-coil region of Phb1 for binding to Phb2. Newly imported Phb1(1-180) formed a cross-link of ~65 kDa (). This adduct was dependent on the presence of Phb2 within mitochondria, and although with low efficiency, could be precipitated with Phb2-specific antibodies (). In addition, we observed the formation of a ~30-kDa cross-link containing Phb1(1-180), which was precipitated with Tim13-specific antibodies (). We conclude from these experiments that the C-terminal coiled-coil region of Phb1 is dispensable for Tim13-binding and the initial interaction with Phb2, whereas it is required for the formation of the large prohibitin complex.
Prohibitins Form Large Ring-shaped Complexes in the Mitochondrial Inner Membrane
The native molecular mass of the assembled prohibitin complex allows to characterize its molecular architecture by electron microscopy. We therefore purified the prohibitin complex from yeast pursuing the following strategies: First, both Phb1 and Phb2 were expressed simultaneously from one multicopy plasmid under the control of galactose-inducible promoters. This resulted in a ~20-fold overexpression of Phb1His and Phb2 compared with wild-type cells (our unpublished data). Second, a hexahistidine peptide was fused to the N terminus of Phb1 (Phb1His) to allow affinity purification by metal chelating chromatography. Expression of Phb1His suppressed the growth defect of a PHB1 deletion in Δyta10 cells, demonstrating the functional activity of Phb1His in vivo (our unpublished data). Notably, overexpressed Phb1His and Phb2 were exclusively detected in mitochondria and not mislocalized to other cellular compartments. The amino-terminal hexahistidine tag did not interfere with the import of Phb1His into mitochondria (our unpublished data). Therefore, isolated mitochondria were used as a starting material for purification. Third, mitochondrial membranes were solubilized in DDM, conditions that destabilize the supercomplex of prohibitins and the m-AAA protease. The prohibitin complex was then purified to homogeneity by metal chelating chromatography and glycerol gradient sedimentation (). Abundant mitochondrial proteins, such as Hsp60, or other membrane-bound protein complexes, such as the F1FO-ATP synthase, bc1 complex, TIM or TOM translocases, m-AAA proteases, or porin were not detected by Western blot analysis in the purified fractions (). Similarly, subunits of the 20S proteasome were immunologically not detectable in the purified protein fraction (our unpublished data).
Figure 6. Single particle electron microscopic analysis of the prohibitin complex. (A) Purification of prohibitin complexes from Δphb1 mitochondria harboring overexpressed Phb1His and Phb2 by metal chelating chromatography and glycerol gradient centrifugation (more ...)
The purified prohibitin complex was investigated by single particle electron microscopy. The mild purification scheme allowed us to detect the majority of particles as single and stable complexes (). Electron micrographs of negatively stained preparations showed various projection views (). Predominant views included roughly elliptical rings with outer dimensions of 270 × 200 Å and a 160 × 90 Å central stain-filled cavity (, classes 1-5) and more rectangular structures frequently composed of two slightly different halves with overall average dimensions of ~260 × 170 Å (, classes 6-10). Similar particles were not detected in control experiments with corresponding fractions from Δphb1Δphb2 cells. Based on these observations, we propose that prohibitins form large ring-like complexes on the inner mitochondrial membrane to which they are anchored by hydrophobic N-terminal helices.