To identify which Tim23p residues are adjacent to specific IMS proteins during import, a single photoreactive probe was incorporated into a site in Tim23p by in vitro translation. When the resulting Tim23p molecules were imported into mitochondria that lacked an IM potential, the photoreactive substrates were trapped in the IMS in complexes with 8-13 and/or 9-10. By using a photoreactive probe, complex formation could be completed before the covalent reaction was initiated with UV light. On illumination (photolysis) of a sample, the activated probe reacted covalently with any nearby protein because the probe's reactivity with different amino acids in target proteins is largely indiscriminate, and the reaction was limited solely to adjacent proteins because the lifetime of the reactive state is very short (nanoseconds). By locating probes at different Tim23p locations, the close juxtaposition of Tim23p to the small Tim proteins was mapped.
To position photoreactive probes in Tim23p, we used an aminoacyl-tRNA analogue approach that we originated (Johnson et al., 1976
; Krieg et al., 1986
). Wild-type Tim23p mRNAs were translated in vitro in the presence of [35
S]Met and εANB-Lys-tRNALys
to yield radioactive Tim23p molecules in which photoreactive εANB-Lys probes replaced ~25% of the lysines incorporated (Krieg et al., 1989
) at each of the 14 lysine codons in Tim23p. Alternatively, to obtain site-specifically labeled Tim23p molecules, Tim23p mRNAs in which a single amber stop codon replaced a natural codon in the sequence were translated as above in the presence of εANB-Lys-tRNAamb
, an amber suppressor tRNA that recognizes an amber stop codon and typically suppresses with an efficiency of 30–50% (Flanagan et al., 2003
; McCormick et al., 2003
; Saksena et al., 2004
; Woolhead et al., 2004
; Davis, unpublished data). The resulting radioactive and photoreactive Tim23p molecules were then imported into mitochondria added to the sample. In most cases reported here, the IM potential was dissipated by CCCP before the mitochondria were added to the sample. After photolysis, the extent of photocross-linking to individual IMS proteins was quantified from the amount of radioactive protein immunoprecipitated by antibodies specific for Tim8p, Tim9p, Tim10p, or Tim13p (Davis et al., 2000
We have previously used this approach to examine nascent chain environment during translation inside the ribosome (Woolhead et al., 2004
), chaperonin-mediated folding (McCallum et al., 2000
; Etchells et al., 2005
), glycosylation (Nilsson et al., 2003
; Karamyshev et al., 2005
), targeting to the membrane (Krieg et al., 1986
; Karamyshev and Johnson, 2005
), and translocation across (Krieg et al., 1989
) and integration into (Thrift et al., 1991
; Do et al., 1996
; McCormick et al., 2003
; Saksena et al., 2004
) the membrane of the endoplasmic reticulum (ER). This approach has also been used successfully in a comprehensive study that identified the TOM and TIM23 complex proteins adjacent to multiple sites in a matrix-directed substrate during import (Kanamori et al., 1997
Photocross-linking of Tim23p and Aac2p to IMS Proteins
To determine whether the photocross-linking approach would work with substrates of the TIM22 pathway, full-length Tim23p and Aac2p were translated in vitro in the presence of [35S]Met and either Lys-tRNALys or εANB-Lys-tRNALys. On incubation with mitochondria, each of these substrates was imported, as evidenced by their presence in the mitochondrial pellet in each sample (p in lanes 3-6 and 9-12 in A). Based on the approximately equal amounts of imported substrate observed in these lanes, the presence of the probe does not inhibit the import of Tim23p or Aac2p significantly, if at all.
The photoreactive probes also survive the import conditions, because photolysis yields radioactive species with higher apparent molecular masses than the precursor protein in the samples containing the ANB probe. When imported into mitochondria lacking an IM potential, both photoreactive Tim23p and Aac2p reacted covalently with proteins with an apparent molecular mass of ~10 kDa (A, lanes 6 and 12). When fully imported into the IM in functional mitochondria, Tim23p reacted covalently with a ~50-kDa protein (A, lane 4), whereas Aac2p did not detectably react with any protein (A, lane 10). Thus, the proteinaceous environments appeared similar for the two TIM22 substrates when their import was arrested in the IMS due to the absence of a ΔΨ, but were different after integration into the IM. Based on previous studies, the likely photocross-linking targets are the small Tim proteins in −ΔΨ mitochondria (Davis et al., 2000
) and Tim50p in the +ΔΨ mitochondria (Geissler et al., 2002
; Yamamoto et al., 2002
); these assignments are confirmed below.
In principle, the absence of a ΔΨ could trap an 8-13ΔTim23pΔ9-10 complex either in the IMS and/or at the TIM22 translocon. Because eliminating the ΔΨ did not cause a detectable formation of a ~45-kDa radioactive species (the expected apparent molecular mass of a Tim23p-Tim22p photoadduct; A), it seems likely that most of the Tim23p molecules are solubilized in the IMS, but we cannot rule out the possibility that the photocross-linked Tim23p in our samples are membrane-bound and arrested at TIM22 (see Discussion).
To identify the ~10-kDa proteins that photocross-link to Tim23p and Aac2p, aliquots of the photolyzed samples containing −ΔΨ mitochondria were immunoprecipitated with antibodies specific for Tim8p, Tim9p, Tim10p, and Tim13p (αTim8p, αTim9p, αTim10p, or αTim13p, respectively). As shown in B, Tim23p reacts covalently with each of these proteins (lanes 3-6; the doublets in the Tim8p and Tim13p lanes are explained below). In contrast, Aac2p photocross-linked only to Tim9p and Tim10p (lanes 9-12). These results are consistent with earlier data (Davis et al., 2000
; Paschen et al., 2000
) showing that only the N-terminal half of a full-length Tim23p chemically cross-links to Tim8p and Tim13p.
To determine whether Tim23p complexes in the IMS are on-pathway or dead-end intermediates, Tim23p was imported into −ΔΨ mitochondria as above. After sedimentation, mitochondria were resuspended either in buffer containing CCCP to maintain the −ΔΨ state or in a buffer containing respiratory substrate and DTT to restore the IM electric potential and Tim23p integration. After photolysis, analysis of the parallel samples revealed that Tim23p photocross-linking to Tim13p was much less in the +ΔΨ than in the −ΔΨ sample (C, compare lanes 1 and 7 with 4 and 10), whereas Tim23p photocross-linking to Tim50p was observed only in +ΔΨ mitochondria (C, compare lanes 5 and 11 with 2 and 8). This marked transition in photocross-linking targets indicates that Tim23p is associated with 8-13 and 9-10 in the IMS in the absence of the ΔΨ, but that restoration of the ΔΨ allows Tim23p to integrate into the IM and associate with Tim50p. The observed changes varied with DTT concentration because DTT facilitates inactivation of CCCP (Yaffe, 1991
) and hence restoration of the ΔΨ, but it also chemically inactivates the photoreactive probe and thereby reduces photoadduct formation. Yet each set of samples showed that IMS-trapped Tim23p was chased into the IM upon restoring its potential. The Tim23p complexes with 8-13 and 9-10 in the absence of ΔΨ are therefore productive intermediates in the TIM22 pathway.
Singly labeled Tim23 Proteins Exhibit Normal Mitochondrial Biogenesis
The above results were obtained with the photoreactive probes distributed randomly in place of Lys residues throughout the primary sequences of Tim23p and Aac2p. To obtain higher resolution information about which residues of Tim23p are adjacent to or contact specific small Tim proteins in the absence of a ΔΨ, photoreactive derivatives of Tim23p were prepared by introducing a single amber stop codon into the Tim23p mRNA coding sequence, initially about every 10 codons in the 222-residue protein (A). In addition, a set of constructs was prepared that had a single amber stop codon at each of 4–6 adjacent codons in TMS 2 and 4. Each modified Tim23p construct was designated TAG-#, where # identifies the wild-type residue substituted with the amber codon and hence the site at which the εANB-Lys probe was incorporated. Translation of these coding sequences in the presence of εANB-Lys-tRNAamb
(Flanagan et al., 2003
; McCormick et al., 2003
; Saksena et al., 2004
) yielded Tim23p proteins with only a single probe located at a defined site.
Figure 2. Tim23p derivatives containing a single εANB-Lys are import competent and form stage-specific photoadducts. (A) Schematic representation of the integrated Tim23p protein and the locations of photoreactive probes. The numbered rectangles indicate (more ...)
When the percentage of amber codon suppression (full-length Tim23p/[full-length Tim23p + Tim23p terminated at the amber codon]) was examined for each of the constructs, we found that the amount of suppression ranged between 20 and 50% for the individual mutants. This suppression efficiency did not correlate with the distance of the amber codon from the initiator codon and instead appeared to vary according to the codon context flanking the amber codon. Evidence for this conclusion is provided by comparing the intensities of the full-length (suppressed) protein bands in B.
It is conceivable that the incorporation of εANB-Lys into Tim23p at a particular position may interfere with its targeting to or import into mitochondria. For example, TAG-131 is located in one of the positively charged loops required for the integration of Tim23p into the IM (Davis et al., 1998
), whereas TAG-156 is located in the middle of the second transmembrane segment where it may affect the ability of Tim23p to fold or insert into the IM. Yet when 35
S-labeled Tim23p, TAG-131, or TAG-156 polypeptides containing εANB-Lys were chemically inactivated by a 10-min, 26°C incubation in 10 mM DTT and then incubated with isolated mitochondria, each was imported across the OM and protected from externally added protease (C, arrowheads, lanes 3, 8, and 13), thereby demonstrating that import across the OM was not compromised by the probe. Furthermore, after disruption of the OM by osmotic shock, a ~12-kDa fragment of Tim23p, diagnostic for its insertion in the IM (Davis et al., 1998
; Ryan et al., 1998
), was generated by protease treatment for each protein (C, lanes 4, 9, and 14). Although these fragments contain fewer [35
S]Met and hence gave less intense phosphorimager bands, the important point is that the extent of import was approximately the same for these three proteins. In contrast, 35
S-labeled Tim23p, TAG-131, or TAG-156 precursors incubated with mitochondria lacking a membrane potential (−ΔΨ) were not inserted into the IM because no ~12-kDa fragment of Tim23p was generated after protease treatment of mitoplasts (C, lanes 5, 10, and 15). Each of the other εANB-Lys–containing Tim23p derivatives was also properly targeted to mitochondria and inserted into the IM (data not shown). Thus, the single εANB-Lys probes incorporated into Tim23p did not interfere with its biogenesis.
Import Stage- and Site-specific Photoadduct Formation
The photocross-linking of singly labeled Tim23p derivatives was examined at two different stages of import: before insertion into the IM (−ΔΨ) and after insertion (+ΔΨ). To distinguish between photoadduct and nonphotoadduct radioactive bands, two samples of each derivative that contained either εANB-Lys-tRNAamb or Lys-tRNAamb were run in parallel. The results of three representative single-probe photocross-linking experiments are shown in D. The non–probe-dependent bands of unknown origin were ignored, and we focused on the probe-dependent high-molecular-mass bands in the gel. For some probe locations (e.g., TAG-25 and TAG-32), photoadducts were observed only when IM insertion was blocked by the absence of a ΔΨ (D, arrowheads, lanes 4 and 8). In other cases (e.g., TAG-66), photoadducts were observed only after insertion into the IM (+ΔΨ; D, asterisk, lane 10), whereas no significant photoadduct formation was observed at other probe locations after IM integration (data not shown). Furthermore, the target proteins differ because the size of the photoadducts are substantially different when probes are located at residue 66 instead of 25 or 32 (compare lanes 4, 8, and 10 in D). These marked differences serve as an important internal control for this approach, because it is clear that the differing photoadduct yields and identities that are both probe- and import stage-specific do not result from some random or systematic photocross-linking, but instead must reflect the local environments of the probes at the time of illumination.
Identification of Site-specific Cross-links to 8-13 and 9-10
To identify residues of Tim23p that are in close proximity to the small Tim proteins, 27 different singly labeled derivatives of Tim23p were individually imported into mitochondria lacking a ΔΨ. After photolysis, a sample was immunoprecipitated either with αTim8p and then αTim9p () or with αTim10p and then αTim13p (). The total sample content of radioactive polypeptide species for each of the entire set of Tim23 single-probe substrates is shown in panel A of both and . These gels reveal a range of high-apparent-molecular-mass photoadducts that contain the photoreactive Tim23p species and target proteins of ~10 kDa (e.g., TAG-25, 32, 80, 90, and 156), ~50 kDa (e.g., TAG-66, 120, and 131), ~70 kDa (e.g., TAG-120, 131, and 181), and ~80 kDa (TAG-80). As is evident by comparing the photoadduct patterns from the two independent sets of samples shown in A and A, the photocross-linking experiments were quite reproducible.
Immunoprecipitations of the samples with small Tim-specific antisera identified which probe sites were able to react covalently with small Tim proteins in the IMS (B and B). An abrupt increase in the apparent molecular mass of Tim23p photoadducts to Tim8p and Tim13p was observed when the probe location moved from residue 80–90 in Tim23p (B and B). This marked change in electrophoretic mobility most likely resulted from a change in the site of covalent linkage between Tim23p and the smaller Tim protein that yielded a different mobility for the photoadduct, as has been observed previously by us (McCormick et al., 2003
) and others (Plath et al., 1998
). A similar effect, photocross-linking of Tim50p from different sites on Tim23p, most likely explains the multiple photoadduct bands in C (lanes 5 and 11). The mobilities of the Tim9p and Tim10p photoadducts did not change.
Quantification of Photocross-linking Efficiences from Different Probe Locations
The efficiency of amber codon suppression differed for the various Tim23p TAG constructs, and hence the amounts of full-length Tim23p produced, imported, and available for photocross-linking varied for the different Tim23p derivatives (compare intensities of “23p” in different lanes of A or A). Thus, to directly compare the efficiencies of covalent reaction from different probe locations to the different small Tim proteins, the gels showing the total imported radioactive protein content in each sample were exposed for the same length of time and to the same phosphorimager plate as the gels that showed the amount of material immunoprecipitated from the same sample. The ratio of photoadduct to unreacted Tim23p was determined from these gels for each Tim23p derivative and small Tim protein. Although this approach (not exposing the gels of the immunoprecipitated material longer) meant that some of the weaker photoadduct bands would not be detected, it provided a reliable and reproducible mechanism for comparing the proximity of individual Tim23p sites to the different small Tim proteins in the IMS.
The results of three independent experiments are shown in . The observed low yields of Tim23p photocross-linking to individual IMS proteins are explained by a combination of effects that includes probe reaction with water or reducing agents, the proximity of probe and target, and inefficient immunoprecipitation. Because of their nucleophilic properties, reducing agents and water react efficiently with electrophilic aryl azides. Hence, photocross-linking efficiencies to nearby proteins are significantly reduced when the probes are in a largely aqueous environment, especially one that contains reducing agents, and are higher when the probes are located in a hydrophobic environment (McCormick et al., 2003
; Saksena et al., 2004
). Hence, when the binding of a probe-carrying molecule to a target protein reduces its exposure to the local aqueous space and/or localizes the probe in close proximity to the target, the chances of the probe reacting covalently with the target before reacting with water increase significantly (e.g., Krieg et al., 1986
). Although the reason(s) for a low photocross-linking efficiency from a particular Tim23p site cannot be conclusively determined from the available data, it would appear that most of the Tim23p probes are in a largely aqueous environment and are not trapped at a protein interface in a Tim23p complex with small Tim proteins, presumably because the probes at the end of the long flexible lysine side chains can be squeezed out of and bend away from such an interface.
Figure 5. Quantification of photoadduct formation. The photoadduct yield (the ratio of Tim23p photoadduct to unreacted Tim23p imported into mitochondria) was determined after immunoprecipitation with αTim8p-, αTim9p-, αTim10p-, or αTim13p-specific (more ...)
Despite the low efficiency of detected covalent reaction, the photocross-linking results reveal a number of important structural features of Tim23p complexes with 8-13 and/or 9-10. Most strikingly, the efficiencies of covalent reaction with a small Tim protein varied tremendously for the 27 different probe locations in Tim23p. If the nonpolar surfaces of the small Tim proteins associated more-or-less randomly with Tim23p only to cover up its hydrophobic surfaces, one would expect to see each probe site in Tim23p photocross-linking to each of the small Tim proteins. Yet we found that each Tim23p residue was reproducibly adjacent only to one and sometimes two small Tim proteins (), suggesting that the interactions of Tim9p, Tim10p, Tim8p, and Tim13p with Tim23p are not random events, and that the various nonpolar regions of Tim23p are not simply being protected from aqueous exposure by relatively nonspecific hydrophobic interactions with nonpolar surfaces on the small Tim proteins acting as nonspecific chaperones.
In general, residues in the N-terminal half of Tim23p photocross-link primarily to Tim8p and Tim13p (B, B, and ), whereas residues in the C-terminal half of Tim23p react covalently primarily with Tim9p and Tim10p (B, B, and ), results consistent with earlier chemical cross-linking data (Davis et al., 2000
; Paschen et al., 2000
). Furthermore, because the photoreactive probe is located at the end of the long, flexible lysine side chain, it is not surprising that a few Tim23p sites are situated where they photocross-linked to more than one small Tim protein. Yet, because some Tim23p sites photocross-linked to only a single small Tim protein, both 8-13 and 9-10 associate with Tim23p in a specific arrangement. For example, Tim13p appears to be in contact primarily with residues 90, 80, and 32, whereas Tim8p is photocross-linked mostly to residue 156 and secondarily to residues 32, 100, 55, and 80 (). Also, although several sites photocross-link to both Tim9p and Tim10p, residue 80 and the sites in TMS4 react almost solely with Tim10p and very little with Tim9p (). We therefore conclude that the small Tim proteins associate with specific sites on the Tim23p substrate to form structurally well-defined complexes.
This conclusion is further supported by comparing the differential photocross-linking observed from each of six consecutive probe locations in the middle of the second hydrophobic TMS (TMS2). Each of the four small Tim proteins is photocross-linked to a residue within this span, but each Tim protein has a distinct reactivity and hence proximity pattern with the six adjacent residues (). To facilitate this comparison, the relative amounts of photocross-linking from these sites to a particular small Tim protein (; residues 153-158) were normalized and are shown in , where the maximum photoadduct yield from these six sites to a given IMS protein was designated as 100%. When the data are expressed in this manner, the resulting histograms clearly show the variations both in photocross-linking to a given IMS protein from the six adjacent sites and also in the proximities of the different IMS proteins to a single site on Tim23p.
Figure 6. Photocross-linking of sites in Tim23p TMS2 to the IMS proteins. The data shown in have been normalized as described in the text to depict the relative photocross-linking efficiencies of probes positioned at residues 153-158 in Tim23p to each (more ...)
For example, the photocross-linking data indicate that Tim8p is primarily adjacent to residue 156 of these six, whereas Tim10p is primarily adjacent to residue 158 and Tim9p to 154 (). Tim13p photocross-linking to these sites is relatively weak compared with its photocross-linking at other sites (), but a reduced covalent reaction was observed at residues 154 and 157 (). Comparing the data of vertically rather than horizontally, residue 158 is exposed primarily to Tim10p, 156 to Tim8p, 155 to Tim13p, and 153 to Tim9p. Although the photocross-linking targets from a particular Tim23p site show some overlap, perhaps because the probe is on a long tether, the distribution of photoadducts is clearly not random.
Photocross-linking Dependence on the N-terminal Half of Tim23p
Tim8p was photocross-linked more efficiently to a probe located in TMS2 than to any other probe, and both Tim8p and Tim13p were photocross-linked to other sites in the C-terminal half of Tim23p (B and ). Although peptide binding experiments indicated that 8-13 associates with the TMSs in Tim23p (Curran et al., 2002b
), previous chemical cross-linking data suggested that 8-13 was only proximal to the N-terminal half of Tim23p and that 9-10 interacted with the C-terminal half (Davis et al., 2000
; Paschen et al., 2000
; Vasiljev et al., 2004
). To clarify the nature of the photoadducts between 8 and 13 and the C-terminus of Tim23p, we repeated the photocross-linking experiments using Tim23Cp, a Tim23p derivative that lacked its N-terminal 94 residues (Ryan et al., 1998
Tim23Cp photocross-linking to Tim9p and Tim10p () was similar to that observed with full-length Tim23p (compare B and B with B, and with C). The association of 9-10 with Tim23p therefore appears to be independent of the N-terminal domain of Tim23p and is mediated almost entirely by interactions between 9 and 10 and the C-terminal half of Tim23p.
In contrast, no photoadducts to Tim8p or Tim13p were detected, even after very long exposures (>4 wk) of the gels to the phosphorimager screens (data not shown). Because no photocross-linking to 8-13 was observed from Tim23Cp residues 155 and 156, it appears that 8-13 does not have significant affinity for binding to the putative TM2 of Tim23Cp. Instead, it seems likely that the proximity of 8-13 to these sites in Tim23p () is dictated by something other than a direct, high-affinity interaction between 8 and 13 and Tim23Cp. We therefore conclude that any positioning of 8-13 adjacent to the C-terminal half of Tim23p is dependent on the presence of the N-terminal half of the protein. Presumably the N-terminal portion of Tim23p binds 8-13 and then folds so as to position 8-13 in close proximity to residues in the C-terminal portion of Tim23p. The three-dimensional arrangement of proteins in the complex may also explain why Tim10p photocross-links with relatively high efficiency to residue 80 and Tim9p to residue 100 in the N-terminal domain of Tim23p ().
Variations in Photocross-linking Due to Putative Substrate Conformation
To what extent are the observed photocross-linking patterns dictated by substrate conformation? Photocross-linking to Tim8p is much higher from position 156 than from either 155 or 157 (), which suggests that this segment of Tim23p is folded in the complex. If Tim23p were unfolded and extended more-or-less linearly at this point, then one would expect the extended, flexible εANB-Lys probe attached at either 155 or 157 to react with significant frequency to Tim8p because the lysine side-chain tether would allow the probes at 155 or 157 in a linear polymer to reach much of the space within reach of a probe at 156. The site specificity of the photocross-linking therefore suggests that TMS2 is folded, perhaps into a helical conformation. If true, probes at 155, 156, and 157 would extend from the helix surface in different directions, and the space accessible to adjacent probes would be reduced. Interestingly, probes at 154 and 157 would be positioned on the same face of the putative helix, and Tim13p is photocross-linked least efficiently from these two sites (). Similarly, Tim10p reacts covalently most efficiently with probes located on the same side of the putative helix, 158 and 155.
Importantly, there are precedents for such variations in photoadduct formation. Major differences in photocross-linking yields and targets have been observed previously with probes positioned at adjacent residues in the α-helices formed by transmembrane sequences during integration into a membrane bilayer at a translocon (McCormick et al., 2003
; Saksena et al., 2004
). Thus, some of the photocross-linking data are consistent with Tim23p being folded when bound to 8-13 and 9-10. However, other experimental approaches will be required to ascertain the actual extent of Tim23p folding in its IMS complex (e.g., Woolhead et al., 2004