The final step in TA protein targeting to the mammalian ER is mediated by TRC40 (also called Asna1)2,4
. This factor interacts directly with TA substrates via their TMD, and upon targeting to the ER membrane via putative receptor(s), releases the TA protein in an ATPase-dependent manner for insertion into the lipid bilayer. The yeast homolog of TRC40, termed Get3, plays an analogous function in conjunction with its ER receptors Get1 and Get2 (3), and still poorly understood cytosolic factors Get4 and Get5 (5-10). In both cases, TRC40/Get3 must selectively and efficiently capture TA proteins via recognition of a hydrophobic TMD that is prone to aggregation, inappropriate interactions, or sequestration by other chaperones.
To investigate how TRC40 efficiently captures its TA protein cargo, we reconstituted this event in vitro
and analyzed its requirements (Sup. Fig. S1
). Sucrose gradient purified ribosome-nascent chains (RNCs) containing a tRNA-tethered TA protein (Sec61β) were released with puromycin in the presence of cytosolic fractions. Capture of the radiolabeled Sec61β by TRC40 was assayed by crosslinking. As expected, Sec61β released into a complete cytosol was captured by TRC40 in a TMD-dependent and energy-stimulated manner. Surprisingly, TRC40 in size fractionated cytosol was unable to capture Sec61β, which instead crosslinked to an unidentified protein we provisionally term p38 (Sup. Fig. S2
). TRC40 capture could be restored by adding back one of the missing fractions. Further fractionation confirmed this ‘capture-stimulating’ factor is distinct from TRC40 (Sup. Fig. S3
). Thus, efficient substrate capture by TRC40 in a complex environment requires an additional protein factor, the absence of which leads to substrate interactions with non-TRC40 proteins (e.g., p38) despite the availability of TRC40.
We postulated the capture-stimulating factor could be an intermediary that delivers substrates to TRC40. Such a factor should recognize TA substrates in a TMD-dependent manner, be found in fractions that contain ‘capture-stimulating’ activity, and be more easily observed upon TRC40 saturation. Crosslinking analyses of Sec61β overproduced in complete cytosol revealed two interacting partners of ~120 kD and 35 kD that met these criteria (Sup. Fig. S4
). Our earlier affinity purification of Sec61β from large-scale translation reactions2
contained an associated product of ~120 kD (in addition to the major TRC40 band) that we identified by mass spectrometry as Bat3 (also known as Scythe/Bag6) (Sup. Fig. S5
). Bat3 affinity purified from reticulocyte lysate co-purified with proteins of ~18 kD and 35 kD () that we identified by mass spectrometry as Ubl4A and C7ORF20, for which we propose the name TRC35 (Sup. Fig. S5
). Antibodies against either Bat3, Ubl4A or TRC35 could co-immunoprecipitate the other two components, and affinity-depletion of any one of these components resulted in substantial depletion of the other two proteins (Sup. Fig. S6
). Thus, Bat3 is part of a stable heterotrimeric complex in the cytosol.
Identification of a TMD-interacting protein complex
The TMD-dependent interaction of the Bat3 complex with TA proteins was verified by immunoblotting of Sec61β affinity purified from in vitro
translation reactions (). Furthermore, the TMD-dependent ~120 kD and 35 kD crosslinks to Sec61β (Sup. Fig. S4
) could be specifically immunoprecipitated under denaturing conditions with anti-Bat3 and anti-TRC35 antibodies, respectively, or under non-denaturing conditions by anti-Ubl4A antibodies (). Analysis of additional TA protein TMDs by crosslinking showed that both TRC40 and the Bat3 complex interacted with the ER-targeted VAMP2 and Sed5 TMDs, while neither interacted with the TMDs from spontaneously inserting cytochrome b5 (Cb5) or mitochondrially-targeted Fis1 or F1L (). Differences in TMD hydrophobicity (either length or maximal hydrophobicity) among these substrates appeared to be a key determinant, since altering Sec61β TMD hydrophobicity to match that of Cb5, Fis1, or F1L markedly reduced interaction with both Bat3 complex and TRC40 (; Sup. Fig. S7
). Thus, the Bat3 complex interacts directly with the TMDs of several ER-targeted TA proteins with similar substrate-specificity as TRC40.
We surmised that the Bat3 complex was responsible for the stimulatory activity needed by TRC40 for efficient substrate capture. In cytosolic fractions immunodepleted (by ~95%) of Bat3 (which also depletes Ubl4A and TRC35 without affecting TRC40 levels; and Sup. Fig. S6
), Sec61β capture by TRC40 is diminished, with a concomitant increase in capture by p38 (); a similar effect is seen after immunodepletion of TRC40, where p38 was the primary soluble interaction partner. Replenishment of Bat3 depleted extracts to the original level with affinity purified Bat3 complex [prepared using an anti-TRC35 antibody (Sup. Fig. S8
)] fully restored TRC40 capture activity (). The effect of Bat3 depletion on TRC40 substrate capture was not simply a consequence of the RNC-release assay since translation of full-length TA substrate in Bat3-depleted reticulocyte lysate also showed diminished TRC40 interactions with increased p38 interaction (Sup. Fig. S9
). Thus, maximally efficient capture by TRC40 of TA proteins upon their release from the ribosome requires the Bat3 complex. These findings are consistent with recent observations suggesting that depletion of Bat3 can impair insertion of a TRC40-dependent TA protein11
The Bat3 complex mediates substrate capture by TRC40
Although the biochemical functions of Bat3, Ubl4A, and TRC35 are poorly understood, the latter two proteins have recognizable homologues in yeast (Mdy2/TMA24/Get5 and Yor164C/Get4, respectively) that have been analysed by recent biochemical, structural, and genetic studies5-7,12,13
. Get4 and Get5 form a stable complex that can interact with Get3, the yeast TRC40 homolog5,7-10,12,13
. Deletion strains of Get4 and Get5 appear to phenocopy Get1, Get2, or Get3 deletions5-7
, and all five genes cluster together in synthetic genetic interaction arrays5,7
. These observations have implicated Get4 and Get5 in TA insertion at a step prior to Get3, but their functions have been unclear.
Given that Bat3 complex facilitates TRC40-substrate interactions, we hypothesized that Bat3 complex might capture TA substrates at the ribosome and transfer them to TRC40. Such a model requires the Bat3 complex to be at or near the ribosome. Indeed, affinity-purified RNCs containing a TA protein were markedly enriched in the Bat3 complex relative to empty ribosomes (). Quantification showed that Bat3 complex and SRP occupy ~27% and ~7%, respectively, of TMD-containing RNCs (). For comparison, SRP occupancy on a genuine SRP substrate isolated by the same method was ~30% (ref. 14
; Sup. Fig. S10
). Notably, Bat3 complex occupancies of <5% were observed for empty ribosomes and RNCs containing a mutant TMD, illustrating substrate selectivity for Bat3 complex recruitment. We confirmed that our affinity purified RNC preparations were tRNA-associated, as judged by their sensitivity to alkaline hydrolysis and selective precipitation with cetyl-trimethyl-ammonium-bromide (CTAB) (Sup. Fig. S11
). Selective enrichment of Bat3 complex on TA protein RNCs was also seen using a magnetic bead ‘pull-up’ strategy that minimizes non-specific recovery of aggregates (Sup. Fig. S12
). Thus, as suggested for SRP14
, the Bat3 complex appears to be recruited to ribosomes when a functional TMD is inside the ribosome.
Bat3 complex captures substrates on ribosomes for transfer to TRC40
Analysis of various TMDs for their capacity to recruit Bat3 complex and SRP to the ribosome from inside the tunnel showed a general correlation with hydrophobicity, with bona fide ER-targeted TMDs (from Sec61β, VAMP2, and Sed5) showing the highest recruitment (Sup. Fig. S10
). However, a strict concordance seems unlikely since a moderately hydrophobic Sec61β mutant (3A3G) that fails to interact with Bat3 complex post-translationally () nonetheless mediated its recruitment from inside the ribosome (Sup. Fig. S10
). Interestingly, Bat3 complex did not crosslink to ribosome-tethered substrate (Sup. Fig. S13
), but only upon release with puromycin or translational termination. This is opposite to SRP, which contacts substrates on the ribosome, but not after release. Thus, ribosomes synthesizing TMD-containing proteins can recruit both SRP and Bat3 complex at a stage before TMD emergence into the cytosol. Both of these complexes stay on the ribosome during further translation (Sup. Fig. S10
), but upon substrate release, only the Bat3 complex remains associated in a TMD-dependent manner. Because changes in SRP recruitment do not influence Bat3 recovery on RNCs, the two factors may not compete with each other. However, this remains to be determined, as the binding site for Bat3 complex is not known.
Selective recruitment of the Bat3 complex to ribosomes before TA protein release suggested this may be the site of initial substrate capture. We therefore looked for a substrate-Bat3-ribosome intermediate upon TRC40 depletion. Sec61β translated in a TRC40-depleted lysate was ~70% ribosome-associated, where it could be crosslinked to Bat3 (). When recombinant TRC40 was included in the depleted translation extract, the proportion of Sec61β in the ribosome fraction was decreased to ~30%, crosslinking to Bat3 was lost, and Sec61β in the soluble fraction was crosslinked to TRC40. Non-ribosomal Sec61β-Bat3 complexes can also transfer substrate to TRC40, since incubation of this fraction with TRC40-containing fractions resulted in a decrease in Bat3 crosslinks and concomitant increase in TRC40 crosslinks (data not shown). Thus, Bat3 complex is recruited to the ribosome, where it can interact with TA substrates upon their translational termination. This putative Bat3-substrate intermediate, whether on the ribosome or free in solution, is then converted into the productive Sec61β-TRC40 targeting complex. Consistent with this model, Bat3 and TRC40 can interact as determined by their co-immunoprecipitation under detergent free conditions ().
Bat3 recruitment to RNCs is restricted to the period between synthesis of the TMD and release from the ribosome (), a time window that seems exceedingly brief. To explain this conundrum, we measured the recruitment window by performing a time course of TA protein synthesis combined with selective precipitation of tRNA-associated polypeptides with CTAB. Nascent chains available for Bat3 complex recruitment would be nearly full-length, but still contain a covalent tRNA. After validating that such tRNA-associated full-length species can be selectively recovered (Sup. Fig. S14
), we quantified the amount of this population during a time course of Sec61β synthesis. Five minutes into this time course (~2 min after full-length Sec61β chains are first detected), a remarkable 50% of full-length Sec61β was precipitated by CTAB (). This proportion diminished over time as translational termination ensued and completed Sec61β chains accumulated. Comparison of experimental data to theoretical expectations (Sup. Fig. S15
) estimate a t1/2
for Sec61β termination of ~ 1 min.
Translation termination is delayed for a TA protein
Remarkably, Sec61β-ΔTMD (lacking the TMD) showed substantially less tRNA-associated polypeptide at each time point () corresponding to a termination t1/2
of ~ 15 sec. Importantly, the last 12 codons (containing an epitope tag) of both constructs are identical, arguing against differences in residues close to the peptidyl transferase centre as the basis for differences in termination rate. The translation elongation rate (~1.5-1.8 sec per residue) and the ~10-fold slower termination rate (relative to elongation) we measured for our control substrate (Sec61β-ΔTMD) are consistent with earlier estimates using this same in vitro system15
. However, the unexpectedly long termination rate of Sec61β suggests that the TMD slows this reaction. The magnitude of termination delay seen for Sec61β (~1 min) is comparable to the elongation ‘arrest’ mediated by SRP measured under similar conditions in this same system16
. This provides a plausible explanation for how Bat3 complex could have sufficient time to be recruited to RNCs containing a TMD inside the tunnel. The sequence parameters that modulate termination remain to be established, but is unlikely to be simple hydrophobicity given earlier studies showing similar termination delays by relatively hydrophilic sequences17
Our findings explain how the hydrophobic TMDs of TA proteins are safely shielded from inappropriate interactions and aggregation during their delivery to the ER membrane (). Our working model posits that the TMD is shielded throughout targeting, first by the ribosome, then by the Bat3 complex, and finally by TRC40. The sequential handoffs of substrate between these complexes are likely to be tightly regulated to prevent exposure of the TMD to aqueous solution. Perhaps the Bat3 complex regulates the nucleotide cycle and/or conformation of TRC40 to facilitate its loading with substrate. Based on recent crystal structures of Get3 (18-22), efficient substrate loading likely requires a nucleotide-dependent transition from an ‘open’ to ‘closed’ conformation18,19
that is perhaps aided by the Bat3 complex. In the absence of this activity, capture by TRC40 would be slower, explaining why it cannot operate efficiently in the presence of competing cytosolic factors, but does manage in a purified system (data not shown).
A role for the Bat3 complex in TA protein targeting may therefore not be absolutely essential23
, but would increase fidelity and efficiency. Such a function would seem to be highly conserved, since yeast have a homologous complex (Get4 and Get5) that shows genetic, physical, and functional interactions with Get35-10,12,13
, and whose absence can lead to aggregation and partial mislocalization of TA proteins5-7
. Thus, the Bat3 complex appears to represent a conserved TMD-selective chaperone that acts at the ribosome. Where on the ribosome the Bat3 complex binds, how it is recruited when a TMD is inside the ribosomal tunnel, and how its function is coordinated with several other ribosome-associating factors including SRP24
, and RAC26
, remain important questions for future studies27