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 First Published: September 24: 2015
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Molecular Biology Arkive Year Alpha

Cell Division: Image: The Institute of Cancer Research, London

 

Molecular Biology Arkive Year Alpha

 

Protein Translocation Channel of Mitochondrial Inner Membrane and Matrix-exposed Import Motor Communicate Via Two-domain Coupling Protein

Rupa Banerjee, Christina Gladkova, Koyeli Mapa, Gregor Witte, Dejana Mokranjac

 

Rupa Banerjee, Christina Gladkova, Koyeli Mapa, Gregor Witte, Dejana Mokranjac (of Ludwig-Maximilians-Universität, Germany) writes in a recently published research in eLife that the majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins.

The function of Tim44 can be rescued by its two domains expressed in trans but not by either of the domains alone. (A) Schematic representation of Tim44 domain structure (numbering according to yeast Tim44 sequence). pre. - presequence (B and C) A haploid yeast deletion strain of TIM44 carrying the wild-type copy of TIM44 on a URA plasmid was transformed with centromeric plasmids carrying indicated constructs of Tim44 under control of endogenous promoter and 3'UTR. Cells were plated on medium containing 5-fluoroorotic acid and incubated at 30°C. The plasmid carrying wild-type Tim44 and an empty plasmid were used as positive and negative controls, respectively. (D) Total cell extracts of wild-type yeast cells transformed with plasmids coding for indicated Tim44 constructs under GPD promoter were analysed by SDS–PAGE and immunoblotting against depicted antibodies. *, ** and *** - protein bands detected with antibodies raised against full-length Tim44.

 

eLife digest

Human, yeast and other eukaryotic cells contain compartments called mitochondria. These compartments are surrounded by two membranes and are most famous for their essential role in supplying the cell with energy. While mitochondria can make a few of their own proteins, the vast majority of mitochondrial proteins are produced elsewhere in the cell and are subsequently imported into mitochondria. During the import process, most proteins need to cross both mitochondrial membranes.

Many mitochondrial proteins are transported across the inner mitochondrial membrane by a molecular machine called the TIM23 complex. The complex forms a channel in the inner membrane and contains an import motor that drives the movement of mitochondrial proteins across the membrane. However, it is not clear how the channel and import motor are coupled together. There is some evidence that a protein within the TIM23 complex called Tim44 – which is made of two sections called the N-terminal domain and the C-terminal domain – is responsible for this coupling. It has been suggested that mainly the N-terminal domain of Tim44 is required for this role.

Banerjee et al. used biochemical techniques to study the role of Tim44 in yeast. The experiments show that both the N-terminal and C-terminal domains are essential for its role in transporting mitochondrial proteins. The N-terminal domain interacts with the import motor, whereas the C-terminal domain interacts with the channel and the mitochondrial proteins that are being moved.

Banerjee et al. propose a model of how the TIM23 complex works, in which the import of proteins into mitochondria is driven by rearrangements in the two domains of Tim44. A future challenge is to understand the nature of these rearrangements and how they are influenced by other components of the TIM23 complex.

Introduction

Mitochondria perform a number of essential cellular functions ranging from production of ATP and diverse other metabolic intermediates to initiation of apoptosis. It is thus not very surprising that disturbances in mitochondrial function are associated with a number of human diseases, including neurodegenerative disorders, diabetes, and various forms of cancer (Nunnari and Suomalainen, 2012; Quirós et al., 2015; Youle and van der Bliek, 2012). An essential prerequisite for correctly functioning mitochondria is import of about 1000 different proteins synthesized as precursor proteins in the cytosol. Recent studies revealed that mitochondrial protein import machineries are sensitive indicators of functionality of mitochondria (Harbauer et al., 2014; Nargund et al., 2012; Yano et al., 2014), demonstrating that a deep understanding of mitochondrial protein import pathways and their regulation will be essential for understanding the role mitochondria have under physiological and pathophysiological conditions. Over half of mitochondrial proteins are synthesized with cleavable, N-terminal extensions called presequences. Import of such precursor proteins requires a coordinated action of the TOM complex in the outer membrane and the TIM23 complex in the inner membrane and is driven by membrane potential across the inner membrane and ATP in the matrix (Dolezal et al., 2006; Endo et al., 2011; Koehler, 2004; Mokranjac and Neupert, 2009; Neupert and Herrmann, 2007; Schulz et al., 2015; Stojanovski et al., 2012).

The TIM23 complex mediates translocation of presequence-containing precursor proteins into the matrix as well as their lateral insertion into the inner membrane. The latter process requires the presence of an additional, lateral insertion signal. After initial recognition on the intermembrane space side of the inner membrane by the receptors of the TIM23 complex, Tim50 and Tim23, precursor proteins are transferred to the translocation channel in the inner membrane in a membrane-potential dependent step (Bajaj et al., 2014Lytovchenko et al., 2013; Mokranjac et al., 2009Shiota et al., 2011Tamura et al., 2009). The translocation channel is formed by membrane-integrated segments of Tim23, together with Tim17 and possibly also Mgr2 (Alder et al., 2008; Demishtein-Zohary et al., 2015leva et al., 2014; Malhotra et al., 2013). At the matrix-face of the inner membrane, precursor proteins are captured by the components of the import motor of the TIM23 complex, also referred to as PAM (presequence translocase-associated motor). Its central component is mtHsp70 whose ATP hydrolysis-driven action fuels translocation of precursor proteins into the matrix (De Los Rios et al., 2006Liu et al., 2003; Neupert and Brunner, 2002Schulz and Rehling, 2014). Multiple cycles of mtHsp70 binding to and release from translocating proteins are required for complete translocation across the inner membrane. The ATP hydrolysis-driven cycling of mtHsp70 and thereby its binding to proteins is regulated by the J- and J-like proteins Tim14(Pam18) and Tim16(Pam16) as well as by the nucleotide-exchange factor Mge1 (D'Silva et al., 2003Kozany et al., 2004Mapa et al., 2010Mokranjac et al., 20062003bTruscott et al., 2003). Tim21 and Pam17 are two nonessential components that bind to Tim17-Tim23 core of the TIM23 complex and appear to modulate its activity in a mutually antagonistic manner (Chacinska et al., 2005Popov-Celeketic et al., 2008van der Laan et al., 2005).

The translocation channel and the import motor of the TIM23 complex are thought to be coupled by Tim44, a peripheral inner membrane protein exposed to the matrix (D'Silva et al., 2004; Kozany et al., 2004; Schulz and Rehling, 2014). Like other components of the TIM23 complex, Tim44 is a highly evolutionary conserved protein and is encoded by an essential gene. In mammals, Tim44 has been implicated in diabetes-associated metabolic and cellular abnormalities (Wada and Kanwar, 1998; Wang et al., 2015). A novel therapeutic approach using gene delivery of Tim44 has recently shown promising results in mouse models of diabetic nephropathy (Zhang et al., 2006). In addition, mutations in Tim44 were identified that predispose carriers to oncocytic thyroid carcinoma (Bonora et al., 2006). Understanding the function of Tim44 and its interactions within the TIM23 complex will therefore be essential for understanding how the energy of ATP hydrolysis is converted into unidirectional transport of proteins into mitochondria and may provide clues for therapeutic treatment of human diseases.

Tim44 binds to the Tim17-Tim23 core of the translocation channel (Kozany et al., 2004; Mokranjac et al., 2003b). Tim44 also binds to mtHsp70, recruiting it to the translocation channel. The interaction between Tim44 and mtHsp70 is regulated both by nucleotides bound to mtHsp70 as well as by translocating proteins (D'Silva et al., 2004; Liu et al., 2003; Slutsky-Leiderman et al., 2007). Tim44 is likewise the major site of recruitment of the Tim14-Tim16 subcomplex, recruiting them both to the translocation channel as well as to mtHsp70 (Kozany et al., 2004; Mokranjac et al., 2003b). In this way, Tim44 likely ensures that binding of mtHsp70 to the translocating polypeptides, regulated by the action of Tim14 and Tim16, takes place right at the outlet of the translocation channel in the inner membrane.

Tim44 is composed of two domains, depicted as N- and C-terminal domains (Figure 1A). Recent studies suggested that the N-terminal domain is responsible for the majority of known functions of Tim44. Segments of the N-terminal domain were identified that are important for interaction of Tim44 with Tim16 and with mtHsp70 (Schilke et al., 2012; Schiller et al., 2008). Furthermore, using site-specific crosslinking, residues in the N-terminal domain were crosslinked to the matrix-exposed loop of Tim23 (Ting et al., 2014). However, the C-terminal domain of Tim44 shows higher evolutionary conservation. Still, the only function that has so far been attributed to the C-terminal domain is its role in recruitment of Tim44 to cardiolipin-containing membranes (Weiss et al., 1999). Based on the crystal structure of the C-terminal domain, a surface-exposed hydrophobic cavity was initially suggested to be important for membrane recruitment (Josyula et al., 2006). However, subsequent biochemical studies combined with molecular dynamics simulations, demonstrated that the helices A1 and A2 (residues 235–262 in yeast Tim44), present in the beginning of the C-terminal domain, are important for membrane recruitment (Marom et al., 2009). Deletion of helices A1 and A2 abolished membrane association of the C-terminal domain. Interestingly, attachment of helices A1 and A2 to a soluble protein was sufficient to recruit it to a model membrane (Marom et al., 2009).

We report here that the function of the full-length Tim44 cannot be rescued by its N-terminal domain extended to include membrane-recruitment helices of the C-terminal domain, demonstrating an unexpected essential function of the core of the C-terminal domain. Surprisingly, we observed that the two domains of Tim44, when expressed in trans, can support, although poorly, growth of yeast cells, giving us a tool to dissect the role of the C-terminal domain in vivo. We identify the C-terminal domain of Tim44 as the domain of Tim44 that is in contact with translocating proteins and that directly interacts with Tim17, a component of the translocation channel. Our data suggest that intricate rearrangements of the two domains of Tim44 are required during transfer of translocating precursor proteins from the channel in the inner membrane to the ATP-dependent motor at the matrix face.

Results

The function of Tim44 can be rescued by its two domains expressed in trans

We reasoned that if all important protein–protein interactions of Tim44 are mediated by its N-terminal domain and the only function of the C-terminal domain is to recruit Tim44 to the membrane, then a construct consisting of the N-terminal domain, extended to include the membrane-recruitment helices A1 and A2, should suffice to support the function of the full-length protein. To test this hypothesis, we cloned such a construct in a yeast expression plasmid and transformed it into a Tim44 plasmid shuffle yeast strain. Upon incubation of transformed cells on a medium containing 5-fluoroorotic acid to remove the URA plasmid carrying the wild-type, full-length copy of Tim44, no viable cells were obtained (Figure 1B). A plasmid carrying the full-length copy of Tim44 enabled growth of yeast cells, whereas no viable colonies were obtained when an empty plasmid was used, confirming the specificity of the assay. We conclude that the N-terminal domain of Tim44, even when extended to include the membrane-recruitment helices of the C-terminal domain, is not sufficient to support the function of the full-length protein. Furthermore, this result suggests that the C-terminal domain of Tim44 has a function beyond membrane recruitment that is apparently essential for viability of yeast cells.

We then tested whether the function of Tim44 can be rescued by its two domains expressed in trans. Two plasmids, each encoding one of the two domains of Tim44 and both including A1 and A2 helices, were co-transformed into a Tim44 plasmid shuffle yeast strain and analyzed as above. Surprisingly, we obtained viable colonies when both domains were expressed in the same cell but not when either of the two domains was expressed on its own (Figure 1C). The rescue was dependent on the presence of A1 and A2 helices on both domains (data not shown), as in their absence neither of the domains could even be stably expressed in yeast (Figure 1D).

It is possible that the two domains of Tim44, both carrying A1 and A2 helices, bind to each other with high affinity and therefore are able to re-establish the full-length protein from the individual domains. To test this possibility, we expressed both domains recombinantly, purified them and analyzed, in a pull down experiment, if they interact with each other. The N-terminally His-tagged N-terminal domain efficiently bound to NiNTA-agarose beads under both low- and high-salt conditions (Figure 1—figure supplement 1A). However, we did not observe any copurification of the non-tagged C-terminal domain. We also did not observe any stable interaction of the two domains when digitonin-solubilized mitochondria containing a His-tagged version of the N-terminal domain were used in a NiNTA pull-down experiment (Figure 1—figure supplement 1B). Thus, the two domains of Tim44 appear not to stably interact with each other.

N+C cells are viable, but grow only very poorly even on fermentable medium

We compared growth rate of the yeast strain carrying the wild-type, full-length version of Tim44 (FL) with that of the strain having two Tim44 domains, both containing A1 and A2 helices, expressed in trans, for simplicity reasons named from here on N+C. The N+C strain was viable and grew relatively well on a fermentable carbon source at 24°C and 30°C (Figure 2A). Still, its growth was slower than that of the FL strain at both temperatures. At 37°C, the N+C strain was barely viable. On a nonfermentable carbon source, when fully functional mitochondria are required, N+C did not grow at any of the temperatures tested. Thus, the function of Tim44 can be reconstituted from its two domains separately, although only very poorly.

We isolated mitochondria from FL and N+C strains grown on fermentable medium and compared their mitochondrial protein profiles. Immunostaining with antibodies raised against full-length Tim44 detected no full-length protein in N+C mitochondria but rather two faster migrating bands (Figure 2B). Based on the running behavior of the individual domains seen in Figure 1D, the slower migrating band corresponds to the N domain and the faster migrating one to the C domain. This confirms that, surprisingly, the full-length Tim44 is indeed not absolutely required for viability of yeast cells. The endogenous levels of other components of the TIM23 complex were either not changed at all (Tim17, Tim23, and Tim50), or were slightly upregulated (mtHsp70, Tim14, and Tim16), likely to compensate for only poorly functional Tim44. Levels of components of other essential mitochondrial protein translocases of the outer and inner mitochondrial membranes, Tom40, Tob55, and Tim22, were not altered compared to FL mitochondria. Similarly, we observed no obvious differences in endogenous levels of proteins present in the outer membrane, intermembrane space, inner membrane, and the matrix that we analyzed.

We conclude that Tim44 can be split into its two domains that are sufficient to support the function of the full-length protein, although only poorly.

Protein import into mitochondria is severely impaired in N+C cells

Considering the essential role of Tim44 during translocation of precursor proteins into mitochondria, we tested whether the severe growth defect of the N+C strain is due to compromised mitochondrial protein import. When import of precursor proteins into mitochondria is impaired, a precursor form of matrix-localized protein Mdj1 accumulates in vivo (Waegemann et al., 2015; Wrobel et al., 2015). We indeed observed a very prominent band of the precursor form of Mdj1 in total cell extracts of N+C cells, grown at 24°C and 30°C, that was absent in cells containing full-length Tim44 (Figure 3A). Thus, the efficiency of protein import into mitochondria is reduced in N+C cells.

To analyze protein import in N+C mitochondria in more detail, we performed in vitro protein import into isolated mitochondria (Figure 3B–G,I–J). To this end, various mitochondrial precursor proteins were synthesized in vitro in the presence of [35S]-methionine and incubated with isolated mitochondria. The import efficiencies of all matrix-targeted precursors analyzed, pF1β, pcytb2(1–167)△DHFR, and pSu9(1–69)DHFR, were drastically reduced in N+C mitochondria when compared to wild type. Import of presequence-containing precursor of Oxa1 that contains multiple transmembrane segments was similarly impaired. Likewise, precursor proteins that are laterally inserted into the inner membrane by the TIM23 complex, such as pDLD1 and pcytb2, were imported with reduced efficiency into N+C mitochondria. In agreement with the established role of Tim44 in import of precursors of a number of components of respiratory chain complexes and their assembly factors, we observed a slightly reduced membrane potential in N+C mitochondria as compared to wild type (Figure 3H). However, precursors of ATP/ADP carrier and of Tim23, whose imports into mitochondria are not dependent on the TIM23 complex, were imported with similar efficiencies in both types of mitochondria, demonstrating that observed effects are not due to general dysfunction of mitochondria. We conclude that splitting of Tim44 into two domains in N+C cells severely impairs transport of proteins by the TIM23 complex, suggesting that full-length Tim44 is required for efficient import of presequence-containing precursor proteins into mitochondria.

Both domains of Tim44 assemble into the TIM23 complex

Tim44 is thought to play an important role in connecting the translocation channel and the import motor of the TIM23 complex. We thus reasoned that disassembly of the TIM23 complex in N+C mitochondria might be a reason for its reduced functionality. When wild-type mitochondria are solubilized with digitonin, affinity-purified antibodies to Tim17 and to Tim23 essentially deplete both Tim17 and Tim23 from the mitochondrial lysate and precipitate part of Tim50, Tim44, Tim14, and Tim16 (Figure 4). Similarly, affinity-purified antibodies to Tim16 deplete both Tim16 and Tim14 and precipitate Tim50, Tim17, Tim23, and Tim44 from mitochondrial lysate. We observed essentially the same precipitation pattern when we analyzed digitonin-solubilized N+C mitochondria, demonstrating that the TIM23 complex is properly assembled. Importantly, both N and C domains of Tim44 were recruited to the TIM23 complex.

The TIM23 complex adopts an altered conformation in N+C mitochondria

Since the assembly of the TIM23 complex is not affected in N+C mitochondria, we reasoned that an altered conformational flexibility may be a reason behind its reduced function in N+C cells. Chemical crosslinking is currently the most sensitive assay available to analyze the conformation of the TIM23 complex in intact mitochondria. We thus compared the crosslinking patterns of TIM23 subunits in N+C mitochondria to those in FL. In wild-type mitochondria, Tim16 can be crosslinked to mtHsp70, Tim44, and Tim14 in an ATP-dependent manner (Figure 5A). In N+C mitochondria, the same crosslinks of Tim16 to mtHsp70 and to Tim14 were observed. The crosslink to Tim44 was, as expected, absent in N+C mitochondria and another crosslink to a smaller protein appeared. In addition, a crosslink between two Tim16 molecules became prominent. Interestingly, this crosslink has previously been observed in mutants in which conformation of the TIM23 complex was altered (Popov-Čeleketić et al., 2008). Similarly, we observed prominent changes in crosslinking pattern of the channel component Tim23 (Figure 5B). In addition to the crosslink of Tim23 to Pam17, observed in both FL and N+C mitochondria, a prominent Tim23-dimer crosslink appeared in N+C mitochondria.

To obtain an independent evidence that the conformation of the TIM23 complex is affected in N+C mitochondria, we analyzed the complex by blue native gel electrophoresis. When digitonin-solubilized wild-type mitochondria are separated by BN-PAGE, Tim17, and Tim23 are present in a 90 kDa complex and, to a lesser degree, in higher molecular weight complexes that additionally contain Tim21 and Mgr2 (Chacinska et al., 2005; Ieva et al., 2014). In contrast, with digitonin-solubilized N+C mitochondria, antibodies to Tim17 and Tim23 revealed slightly shifted bands, in particular of the 90 kDa complex (Figure 5C). Since the 90 kDa complex does not contain any other known subunit of the TIM23 complex, this finding further supports the above notion that the conformation of the translocation channel is changed in N+C mitochondria. We observed no obvious difference in the ca. 60 kDa Tim14-Tim16 complex between FL and N+C mitochondria. As expected, full-length Tim44, present in FL mitochondria, was absent in N+C mitochondria (Figure 5C).

Together, these results demonstrate that the conformation of the TIM23 complex is changed in N+C mitochondria. They further show that alterations in the components traditionally assigned to the import motor affect the conformation of the translocation channel in the inner membrane, supporting the notion of an intricate crosstalk within the complex.

Role of the C-terminal domain of Tim44

The data presented so far suggest that full-length Tim44 is required for optimal conformational dynamics of the TIM23 complex. Furthermore, they suggest that the C-terminal domain has an essential function within the TIM23 complex, beyond mere membrane recruitment. So, what is the function of the C-terminal domain of Tim44? We first searched for binding partners of the individual domains. To that end, we recombinantly expressed and purified full-length Tim44 as well as its two domains (Figure 6A). To look for interaction partners of the core domains, both domains now lacked the segment containing A1 and A2 helices. Purified proteins were covalently coupled to the Sepharose beads and were subsequently incubated with mitochondrial lysates. Mitochondria were solubilized with Triton X-100 that, unlike digitonin, dissociates the TIM23 complex into its individual subunits (except for the Tim14-Tim16 subcomplex that remains stable). In this way, direct protein-protein interactions can be analyzed. We observed prominent, specific binding of mtHsp70, Tim16, Tim14 and Tim17, and to a far lesser degree of Tim23 and Tim50, to full-length Tim44 (Figure 6B). None of the proteins bound to empty beads. Also, we observed no binding of two abundant mitochondrial proteins, porin, and F1βß, demonstrating the specificity of observed interactions. mtHsp70, Tim16 and Tim14 also efficiently bound to the N-terminal domain of Tim44, in agreement with previous observations (Schilke et al., 2012; Schiller et al., 2008), and far less efficiently to the C-terminal domain. Since the Tim14-Tim16 subcomplex remains stable in Triton X-100, it is not possible by this method to distinguish which of the two subunits, or maybe even both, directly interacts with the N-terminal domain of Tim44. Binding of Tim17 to the N-terminal domain of Tim44 was drastically lower compared to its binding to the full-length protein. Instead, a strong binding of Tim17 to the C-terminal domain of Tim44 was observed.

We conclude that the N-terminal domain of Tim44 binds to the components of the import motor, whereas the C-terminal domain binds to the translocation channel in the inner membrane, revealing a novel function of the C-terminal domain of Tim44.

We then asked which of the two domains of Tim44 is in contact with translocating proteins. To answer this question, we first affinity-purified antibodies that specifically recognize cores of the individual domains of Tim44 using the above described Sepharose beads. The antibodies, affinity purified using beads with coupled full-length Tim44, recognized full-length Tim44 as well as both of its domains (Figure 6C). In contrast, antibodies that were affinity purified using beads with coupled individual domains recognized only the respective domain and the full-length protein (Figure 6C). This demonstrates that we indeed purified antibodies specific for individual domains of Tim44. Next, we accumulated 35S-labelled precursor protein pcytb2(1–167)△DHFR as a TOM-TIM23-spanning intermediate. Briefly, this precursor protein consists of the first 167 residues of yeast cytochrome b2, with a 19 residue deletion in its lateral insertion signal, fused to the passenger protein dihydrofolate reductase. In the presence of methotrexate, that stabilizes folded DHFR, the b2 part reaches the matrix, whereas the DHFR moiety remains on the mitochondrial surface resulting in an intermediate that spans both TOM and TIM23 complexes. The association of Tim44 and its domains with the arrested precursor protein was analyzed by chemical crosslinking followed by immunoprecipitation with antibodies to full-length Tim44 and its individual domains. In wild-type mitochondria, all three antibodies precipitated a crosslinking adduct of Tim44 to the arrested precursor protein, demonstrating that they are all able to immunoprecipitate the respective antigens (Figure 6D). In contrast, with N+C mitochondria, a faster migrating crosslinking adduct of a Tim44 domain to the arrested precursor protein was immunoprecipitated with the antibodies against the C-terminal domain and against the full-length protein but not with the antibodies against the N-terminal domain. This demonstrates that the C-terminal domain of Tim44 is in close vicinity of the translocating protein.

Mutations identified in human patients can frequently point to functionally important residues in affected proteins. In this respect, Pro308Gln mutation in human Tim44 has recently been linked to oncocytic thyroid carcinoma (Bonora et al., 2006). Since the mutation maps to the C-terminal domain of Tim44, we wanted to analyze functional implications of this mutation and therefore made the corresponding mutation in yeast Tim44 (Pro282Gln). We compared thermal stabilities of wild type and mutant Tim44 proteins by thermal shift assay. The melting temperature of wild-type Tim44 was 54°C, whereas that of the mutant protein was 4°C lower (Figure 6E). This demonstrates that the mutation significantly destabilizes Tim44, providing first clues toward molecular understanding of the associated human disease.

Discussion

The major question of protein import into mitochondria that has remained unresolved is how translocation of precursor proteins through the channel in the inner membrane is coupled to the ATP-dependent activity of the Hsp70-based import motor at the matrix face of the inner membrane.

Results presented here demonstrate that the two domain structure of Tim44 is essential during this process. We show here that the two domains of Tim44 have different interaction partners within the TIM23 complex. In this way, Tim44 holds the TIM23 complex together. Our data revealed a direct, previously unexpected interaction between the C-terminal domain of Tim44 with the channel component Tim17. This result not only assigned a novel function to the C-terminal domain of Tim44 but also shed new light on Tim17, the component of the TIM23 complex that has been notoriously difficult to analyze. Recent mutational analysis of the matrix exposed loop between transmembrane segments 1 and 2 of Tim17 revealed no interaction site for Tim44 (Ting et al., 2014), suggesting its presence in another segment of the protein. Our data also confirmed the previously observed interactions of the N-terminal domain of Tim44 with the components of the import motor (Schilke et al., 2012; Schiller et al., 2008). We did, however, not observe any direct interaction between Tim23 and the N-terminal domain of Tim44 that has previously been seen by crosslinking in intact mitochondria (Ting et al., 2014). It is possible that this crosslinking requires a specific conformation of Tim23 only adopted when Tim23 is bound to Tim17 in the inner membrane. This notion is supported by our previous observation that the stable binding of Tim44 to the translocation channel requires assembled Tim17-Tim23 core of the TIM23 complex (Mokranjac et al., 2003b). We observed a direct Tim17-Tim44 interaction here probably because of a high local concentration of the C-terminal domain when bound to the beads.

The core of the C-terminal domain is preceded by a segment that contains two amphipathic, membrane-recruitment helices. This central segment connects the two domains of Tim44. Intriguingly, the two currently available crystal structures of the C-terminal domains of yeast and human Tim44s showed different orientations of the two helices relative to the core domains (Handa et al., 2007; Josyula et al., 2006). The conformational change was likely induced upon PEG binding to this region of human Tim44 during crystallization (Handa et al., 2007). It is tempting to speculate that the same conformational change takes place during translocation of proteins in the mitochondria. Such a conformational change would not only reorient the two helices in respect to the core of the C-domain but also change the relative orientation of N- and C-terminal domains. Since the two domains have different interaction partners within the TIM23 complex, such a change could rearrange the entire complex. The importance of this proposed conformational change in Tim44 is supported by the data presented here. The function of the full-length Tim44 could be reconstituted from its individual domains only very poorly. Also, there is obviously a very strong evolutionary pressure to keep the two domains of Tim44 within one polypeptide chain. N+C strain had to be kept at all times on the selective medium - even after only an overnight incubation on a nonselective medium the full-length protein reappeared (our unpublished observation), likely due to a recombination event between two plasmids.

Tim44 can be crosslinked to translocating proteins. Our data revealed that it is the C-terminal domain of Tim44 that interacts with proteins entering the matrix from the translocation channel in the inner membrane. A direct interaction of the same domain with Tim17 would optimally position the C-terminal domain to the outlet of the translocation channel. This raises an interesting possibility that translocating precursor proteins may play an important role in the above postulated conformational changes of Tim44.

A missense mutation Pro308Gln in human Tim44 is associated with familial oncocytic thyroid carcinoma. The corresponding mutation in yeast, Pro282Gln, destabilized the protein but produced no obvious growth phenotype or an in vivo import defect (our unpublished observations), suggesting that the yeast system is more robust. This observation is in agreement with the notion that mutations that would severely affect the function of the TIM23 complex would likely be embryonically lethal in humans. Still, the disease caused by a mutation in the C-terminal domain of human Tim44 speaks for an important role of this domain in the function of the entire TIM23 complex. Furthermore, the mutation maps to the short loop between A3 and A4 helices in the C-terminal domain of Tim44. Based on the crystal structure of Tim44, it was previously suggested that the mutation could affect the conformational flexibility of the A1 and A2 helices (Handa et al., 2007), intriguingly providing further support for the above postulated conformational changes of Tim44.

Based on the previously available data and the results presented here, we put forward the following model to describe how translocation of precursor proteins through the channel in the inner membrane is coupled to their capture by the ATP-dependent import motor at the matrix face of the channel (Figure 7). Tim44 plays a central role in this model. We envisage that two domains of Tim44 are connected by the central segment that contains membrane-recruitment helices, like two cherries on the stalks (Figure 7 insert). This central segment of Tim44 recruits the protein to the cardiolipin-containing membranes. There, through direct protein–protein interactions, the C-terminal domain of Tim44 binds to Tim17 and the N-terminal domain to mtHsp70 and to Tim14-Tim16 subcomplex (1). In this way, Tim44 functions as a central platform that connects the translocation channel in the inner membrane with the import motor at the matrix face. Additional interactions likely stabilize the complex, in particular that between the N-terminal domain of Tim44 and Tim23 (Ting et al., 2014) as well as the one between Tim17 and the IMS-exposed segment of Tim14 (Chacinska et al., 2005). In the resting state, the translocation channel is closed to maintain the permeability barrier of the inner membrane. During translocation of proteins (2), the translocation channel in the inner membrane has to open to allow passage of proteins. Opening of the channel will likely change the conformation of Tim17 that could be further conveyed to the C-terminal domain Tim44. It is tempting to speculate that this conformational change is transduced to the N-terminal domain of Tim44 through the central, membrane-bound segment of Tim44, leading to relative rearrangements of the two domains of Tim44. This change would now allow Tim14-Tim16 complex to stimulate the ATPase activity of mtHsp70 leading to stable binding of the translocating protein to mtHsp70. mtHsp70, with bound polypeptide, will then move into the matrix, opening a binding site on Tim44 for another molecule of mtHsp70 (3). We speculate that the release of mtHsp70 with bound polypeptide from the N-terminal domain of Tim44 will send a signal back to the C-terminal domain of Tim44 and further to the translocation channel. Multiple cycles of mtHsp70 are required to translocate the entire polypeptide chain into the matrix. Once the entire polypeptide has been translocated, the translocation channel will revert to its resting, closed state, bringing also Tim44 back to its resting conformation (1). Thus, the translocation channel in the inner membrane and the mtHsp70 system at the matrix face communicate with each other through rearrangements of the two domains of Tim44 that are stimulated by translocating polypeptide chain.

Material and methods

Yeast strains, plasmids, and growth conditions

Wild-type haploid yeast strain YPH499 was used for all genetic manipulations. A Tim44 plasmid shuffling yeast strain was made by transforming YPH499 cells with a pVT-102U plasmid (URA marker) containing a full-length TIM44 followed by replacement of the chromosomal copy of TIM44 with a HIS3 cassette by homologous recombination. For complementation analyzes, endogenous promoter, mitochondrial presequence (residues 1–42) and the 3’-untranslated region of TIM44 were cloned into centromeric yeast plasmids pRS315 (LEU marker) and pRS314 (TRP marker) and obtained plasmids subsequently used for cloning of various Tim44 constructs. The following constructs were used in the analyzes: Tim44(43–209), Tim44(43–262), Tim44(264–431), and Tim44(210–431). The constructs encompassing the N- and the C-terminal domains of Tim44 were cloned into pRS315 and pRS314 plasmids, respectively. Plasmids carrying the full-length copy of TIM44 were used as positive controls and empty plasmids as negative ones. A Tim44 plasmid shuffling yeast strain was transformed with two plasmids simultaneously and selected on selective glucose medium lacking respective markers. Cells that lost the wild-type copy of Tim44 on the URA plasmid were selected on medium containing 5-fluoroorotic acid at 30°C.

For expression in the wild-type background, the above-described constructs of Tim44, containing endogenous Tim44 presequence, were also cloned into centromeric yeast plasmids p414GPD and p415GPD for expression under the control of the strong GPD promoter. Cells were grown on selective lactate medium containing 0.1% glucose.

FL and N+C cells were grown in selective glucose medium at 30°C, unless otherwise indicated, and mitochondria were isolated from cells in logarithmic growth phase.

Recombinant proteins

DNA sequences coding for various segments of Tim44 were cloned into bacterial expression vector pET-Duet1 introducing a TEV cleavage site between the His6-tag and the protein coding region. The following Tim44 constructs were cloned: Tim44(43–431) (full-length protein lacking the mitochondrial presequence), Tim44(43–209) (referred to as N in Figure 6A), Tim44(43–263), Tim44(211–431), and Tim44(264–431) (referred to as Cc in Figure 6A). Pro282Gln mutation was introduced into the full-length construct using site directed mutagenesis. Proteins were expressed in E. coli BL21(DE3) at 37°C and purified using affinity chromatography on NiNTA-agarose beads (Qiagen, Germany) followed by gel filtration on Superdex 75 column (GE Healthcare, Germany). Unless otherwise indicated, the His6-tags were removed by incubation with the TEV protease. The purified proteins were stored at -80oC in 20 mM HEPES/KOH, 200 mM KCl, 5 mM MgCl2, pH 7.5, until use.

Purified proteins were coupled to CNBr-Sepharose beads (GE Healthcare, Germany) according to manufacturer's instructions and stored at 4°C. The beads were used for purification of domain-specific antibodies from the serum raised in rabbits against recombinantly expressed full-length Tim44. For direct binding analysis, mitochondria isolated from wild-type yeast cells were solubilized with 0.5% Triton X-100 in 20 mM Tris/HCl, pH 8.0, 80 mM KCl, 10% glycerol at 1 mg/mL and incubated with Tim44 constructs coupled to CNBr-Sepharose beads for 30 min at 4oC. After three washing steps, specifically bound proteins were eluted with Laemmli buffer. Samples were analyzed by SDS–PAGE and immunoblotting.

Figure 7. A proposed model of function of the TIM23 complex. See text for details. For simplicity reasons, only essential subunits of the complex are shown.

Thermal shift assay

Thermal stabilities of wild type and P282Q mutant form of Tim44 were analyzed by fluorescence thermal shift assay (Müller et al., 2015). Recombinant proteins (6.2 µM) in 20 mM HEPES/NaOH, 150 mM NaCl, pH 7.1 were mixed with 5x SYPRO Orange and melting curves analyzed in a real-time PCR machine using a gradient from 5°C to 99°C. Three technical replicates of two independent protein purifications were analyzed in parallel. Mutant Tim44 showed significantly decreased thermal stability under all conditions analyzed - in buffers containing different salt concentrations (50, 150, and 450 mM) as well as in different buffers and pHs (HEPES buffer at pH 7.1 and phosphate buffer at pH 8.0).

Miscellaneous

Previously published procedures were used for protein import into isolated mitochondria, crosslinking, coimmunoprecipitations and arrest of mitochondrial precursor proteins as TOM-TIM23 spanning intermediates followed by crosslinking and immunoprecipitation under denaturing conditions (Mokranjac et al., 2003a; 2003b; Popov-Čeleketić et al., 2008).

The Article can be read in full in eLife DOI

( Published December 29, 2015 Cite as eLife 2015;4:e11897)

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Fibroblastic Reticular Cell-derived Lysophosphatidic Acid Regulates Confined Intranodal T-cell Motility
 

Akira Takeda
Daichi Kobayashi
Keita Aoi
Naoko Sasaki
Yuki Sugiura
Hidemitsu Igarashi
Kazuo Tohya
Asuka Inoue
Erina Hata
Noriyuki Akahoshi
Haruko Hayasaka
Junichi Kikuta
Elke Scandella
Burkhard Ludewig
Satoshi Ishii
Junken Aoki
Makoto Suematsu
Masaru Ishii
Kiyoshi Takeda
Sirpa Jalkanen
Masayuki Miyasaka
Eiji Umemoto

In a recently published research in eLife the above named authors write that lymph nodes (LNs) are highly confined environments with a cell-dense three-dimensional meshwork, in which lymphocyte migration is regulated by intracellular contractile proteins. However, the molecular cues directing intranodal cell migration remain poorly characterized. Here we demonstrate that lysophosphatidic acid (LPA) produced by LN fibroblastic reticular cells (FRCs) acts locally to LPA2 to induce T-cell motility. In vivo, either specific ablation of LPA-producing ectoenzyme autotaxin in FRCs or LPA2 deficiency in T cells markedly decreased intranodal T cell motility, and FRC-derived LPA critically affected the LPA2-dependent T-cell motility. In vitro, LPA activated the small GTPase RhoA in T cells and limited T-cell adhesion to the underlying substrate via LPA2. The LPA-LPA2 axis also enhanced T-cell migration through narrow pores in a three-dimensional environment, in a ROCK-myosin II-dependent manner. These results strongly suggest that FRC-derived LPA serves as a cell-extrinsic factor that optimizes T-cell movement through the densely packed LN reticular network.

eLife digest

Small organs called lymph nodes are found throughout the body and help to filter out harmful particles and cells. Lymph nodes are packed with different types of immune cells, such as the T-cells that play a number of roles in detecting and destroying bacteria, viruses and other disease-causing microbes. Within the lymph node, T-cells crawl along a meshwork made up of cells called fibroblastic reticular cells. The T-cells appear to move in random patterns, but the signals that drive this movement remain ill-defined.

Now, Takeda et al. reveal that a lipid called lysophosphatidic acid (LPA), which is produced by the fibroblastic reticular cells, is responsible for regulating how T-cells move around inside the lymph nodes. T-cells are able to detect LPA via certain receptor proteins on their surface. Takeda et al. engineered mice that were either unable to produce a particular LPA receptor on their T-cells, or that produced less LPA than normal. The T-cells of these mice moved around less than T-cells in normal mice.

Further experiments revealed that LPA signaling also affects the signaling pathway that alters how well the T-cells stick to nearby surfaces. This suggests that LPA helps to optimize T-cell movement to allow the cells to navigate the small spaces found between the fibroblastic reticular cells. In the future, targeting the processes involved in LPA signaling could help to develop new treatments for disorders of the immune system.

Introduction

Blood-borne naïve lymphocytes migrate along the fibroblastic reticular cell (FRC) network in lymph nodes (LNs) (von Andrian and Mempel, 2003; Miyasaka and Tanaka, 2004; Girard et al., 2012). B cells then migrate into LN follicles, whereas T cells remain in the paracortex and migrate continually along the FRC network (Bajénoff et al., 2006). This intranodal migration provides critical opportunities for T cells to encounter cognate antigen-presenting dendritic cells. Two-photon microscopic analysis has shown that naïve T cells crawl along the FRC network in an apparently random pattern of motion, at an average velocity of 10–15 μm per minute (Miller et al., 2002; Okada and Cyster, 2007; Worbs et al., 2007). FRCs promote intranodal T-cell motility by signaling naïve lymphocytes with CCL21/CCL19 via CCR7, thus activating the small GTPase Rac (Okada and Cyster, 2007; Worbs et al., 2007; Faroudi et al., 2010; Huang et al., 2007), although CCR7 signaling only partially account for the interstitial T cell motility (Okada and Cyster, 2007; Huang et al., 2007).

LPA is a bioactive lysophospholipid produced both extracellularly and intracellularly. Extracellularly produced LPA is involved in such diverse biological functions as vascular remodeling and cell growth, survival, and migration (Choi et al., 2010; Yanagida et al., 2013). Intracellularly produced LPA is an intermediate in the synthesis of triglycerides and glycerophospholipids, and thought to act as a 'housekeeper' inside the cell (Mills and Moolenaar, 2003). Extracellular LPA is predominantly produced by autotaxin (ATX, also referred to as ENPP2 [ectonucleotide pyrophosphatase/phosphodiesterase family member 2]), an ectoenzyme that was originally identified as a tumor-cell motility-enhancing factor (Stracke et al., 1992). ATX is a lysophospholipase D that produces LPA by hydrolyzing lysophosphatidylcholine (LPC) (Okudaira et al., 2010; Moolenaar and Perrakis, 2011). We and others have reported that ATX is strongly expressed in HEV endothelial cells (ECs), and that ATX regulates lymphocyte migration into the LN parenchyma (Kanda et al., 2008; Nakasaki et al., 2008; Umemoto et al., 2011). We also demonstrated that LPA enhances lymphocyte detachment from ECs and promotes lymphocyte transmigration across the HEV basal lamina, at least in part by acting on HEV ECs (Bai et al., 2013). LPA also acts on naïve T cells to induce chemokinesis and cell polarization (Kanda et al., 2008; Katakai et al., 2014; Zhang et al., 2012) and transmigration (Zhang et al., 2012).While a study using pharmacological inhibitors revealed that ATX/LPA promotes intranodal lymphocyte motility in an ex vivo LN explant model (Katakai et al., 2014), the physiological significance of the ATX/LPA axis in interstitial lymphocyte migration remains unknown.

To date, six LPA receptors (LPA1–LPA6) have been identified. LPA receptors couple to multiple G proteins, including Gi, G12/13, Gq, and Gs, and upon ligand binding, these G proteins activate diverse intracellular signaling components including Rho and Rac. Although LPA2 has recently been reported to play a role in intranodal T-cell migration (Knowlden et al., 2014), it remains unclear how LPA2-mediated signaling affects interstitial T-cell motility and whether LPA is the prime activating ligand.

Leukocyte migration in a confined environment is regulated at least partly by cell contraction (Lämmermann et al., 2008). Jacobelli et al. (2010) reported that a contractile protein, myosin IIA, is required for T-cell amoeboid motility in confined environments such as LNs (Jacobelli et al., 2010). Myosin IIA cross-links actin, thus limiting surface adhesion and allowing T cells to exert contractile force. Myosin IIA’s activity is regulated by RhoA and Rho-associated protein kinase (ROCK) signaling. While the cell-extrinsic factor(s) that regulate myosin II’s activity during T-cell migration in a confined environment have been poorly defined, a recent study using zebrafish germ progenitor cells showed that LPA induces cell polarization in a ROCK-myosin II-dependent manner, which enables rapid cell migration in a confined environment (Ruprecht et al., 2015).

In this study, by using mice conditionally deficient for the LPA-generating enzyme ATX in FRCs and those deficient in LPA2, we demonstrated that bioactive LPA species are produced by FRCs in an ATX-dependent manner and that LPA acts locally on LPA2 on T cells. This LPA2-mediated signaling activates the RhoA-ROCK-myosin II pathway and promotes confinement-optimized interstitial T-cell migration. The FRC-derived LPA thus serves as a cell-extrinsic factor that optimizes T-cell movement through the densely packed LN reticular network, to fine-tune T-cell trafficking.

Results

FRCs express the LPA-generating enzyme ATX in a lymphotoxin β receptor (LTβR)-signaling-dependent manner

Although CCR7 ligands are reported to stimulate intranodal T-cell motility (Okada and Cyster, 2007; Worbs et al., 2007; Huang et al., 2007), they are not sufficient to account for effective T-cell migration in LNs. Given that ATX, which generates the motogenic lysophospholipid, LPA, is expressed in HEV ECs to modulate lymphocyte motility (Nakasaki et al., 2008; Bai et al., 2013), we speculated that ATX and its product LPA are also expressed in other stromal cell subsets in the LN parenchyma and may control intranodal lymphocyte migration. Indeed, a recent paper showed that ATX is expressed in CCL21+ CD31- stromal cells in LNs (Katakai et al., 2014). We therefore subdivided the CD45- LN stromal cells into four stromal subsets (Figure 1A). As shown in Figure 1B, Enpp2/Autotaxin was readily detected in GP38+ CD31- FRCs as well as GP38- CD31+ blood endothelial cells (ECs), with negligible expression in lymphatic ECs and double-negative cells. Electron microscopic analysis confirmed that ATX was expressed in the FRCs surrounding collagen fiber bundles (Figure 1C). Interestingly, analyses using Ccl19-Cre x R26-EYFP mice, which constitutively express yellow fluorescent protein (YFP) in FRCs (Chai et al., 2013), revealed that Enpp2 was selectively expressed in LN FRCs but not splenic FRCs (Figure 1D). This Enpp2 expression was apparently dependent on LTβR signaling, because blocking LTβR signaling significantly reduced expression of Enpp2, Cxcl13, and Ccl19, but not Icam1, in FRCs (Figure 1E). This blockade also downregulated transcription of HEV marker genes such as Glycam1 and Ccl21 in BECs (Figure 1E), consistent with a previous report (Browning et al., 2005). These results confirm that similarly to HEV ECs, FRCs constitutively express the LPA-generating enzyme ATX, which is maintained at least in part by LTβR signaling.

Multiple LPA species are produced in the LN parenchyma by FRCs

To verify that LPA is generated in situ by FRC-derived ATX, we crossed Enpp2fl/fl mice and Ccl19-Cre mice to generate Ccl19-Cre Enpp2fl/fl mice that lacked ATX expression specifically in the FRCs. As expected, in the Ccl19-Cre Enpp2fl/fl mice, Enpp2 was completely lost in the FRCs but not in the BECs, whereas Ccl21 and GP38 expression was comparable between these strains (Figure 2A, Figure 2—figure supplement 1). The frequency of FRCs in stromal cells also appeared to be uncompromised by the deficiency of Enpp2 in FRCs (Figure 2—figure supplement 1). We then compared LPA production in the LN of these mice using imaging mass spectrometry (IMS). To this end, we first injected fluorescein-conjugated dextran, which labels lymphatics and the medulla, into the footpad, and LPA (18:0), LPA (18:1), LPA (18:2), and LPA (20:4) were then visualized in LN sections. As shown in Figure 2B, signals corresponding to LPA (18:0) were widely distributed in the LN. The signals were comparable in intensity and frequency in Enpp2fl/fl and Ccl19-Cre Enpp2fl/fl mice; this LPA species appears to be produced mainly within the cell (Aoki, J; unpublished observation) independently of ATX (Yukiura et al., 2011, Nishimasu, et al., 2011). In sharp contrast, signals corresponding to LPA (18:1), LPA (18:2), and LPA (20:4), the major species produced extracellularly by ATX (Yukiura et al., 2011), were predominantly observed in the paracortex both close to and at a distance from HEVs, but only marginally in the medulla. These signals were substantially decreased in the cortex of Ccl19-Cre Enpp2fl/fl as compared with Enpp2fl/fl mice (Figure 2B).

To verify that the cortical LPA signals associated with non-HEV structures were derived from FRCs, we next mapped the LPA signals relative to HEVs in Enpp2fl/fl mice and Ccl19-Cre Enpp2fl/fl mice by measuring the distance between individual signals and the nearest HEV. As shown in Figure 2C, the frequency of LPA (18:1), LPA (18:2), and LPA (20:4) signals within 50 μm of an HEV did not differ significantly between Enpp2fl/fl and Ccl19-Cre Enpp2fl/fl mice. However, the frequency of relatively distant signals (more than 50 μm) decreased substantially when ATX was ablated in FRCs. Hence, the median distance between LPA signals and HEVs was significantly reduced in Ccl19-Cre Enpp2fl/fl compared with Enpp2fl/fl mice, consistent with the idea that distant LPA signals were associated with FRCs. Together with the observations showing robust expression of ATX in FRCs, these findings indicate that the cortical LPA signals not associated with HEVs are mainly produced by FRCs in an ATX-dependent manner.

FRC-derived LPA promotes intranodal T-cell migration

To understand the role of FRC-derived LPA in regulating intranodal T-cell migration, we next examined the CD4+ T-cell interstitial migration in LNs by intravital two-photon microscopy. To this end, CD4+ T cells from WT mice expressing a transgene encoding enhanced GFP (eGFP) were injected intravenously into Enpp2fl/fl and Ccl19-Cre Enpp2fl/fl mice, and the intranodal T-cell migration in popliteal LNs (PLNs) was imaged 15–25 hr later (Video 1). As shown in Figure 3A, B, CD4+ T-cell movement and displacement from the original location in the PLN was substantially restricted in Ccl19-Cre Enpp2fl/fl compared with Enpp2fl/fl mice. The median T-cell velocity was also lower in Ccl19-Cre Enpp2fl/fl than in Enpp2fl/fl mice (Figure 3C, Figure 3—figure supplement 1). Measurement of the mean displacement and the motility coefficient, which represents the volume in which an average cell scans per unit time (Sumen et al., 2004), also indicated that T-cell motility was impaired in the LN parenchyma of Ccl19-Cre Enpp2fl/fl mice (Figure 3D,E), whereas the directionality of the intranodal T-cell movement was comparable in these mouse groups (Figure 3—figure supplement 2), supporting the hypothesis that FRC-derived LPA is required for efficient intranodal T-cell migration.

The Article can be read in full in eLife DOI

( The Authors are from: Osaka University Graduate School of Medicine, Japan; Osaka University, Japan; University of Turku, Finland; Keio University School of Medicine, Japan; JST Precursory Research for Embryonic Science and Technology project, Japan; Akita University, Japan; Kansai University of Health Sciences, Japan; Tohoku University, Japan; Kantonal Hospital St. Gallen, Switzerland; Core Research for Evolutional Science and Technology project, Japan)

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Attenuation of AMPK Signaling by ROQUIN Promotes T Follicular Helper Cell Formation


In a recent research published in eLife Roybel R Ramiscal, Ian A Parish, Robert S Lee-Young, Jeffrey J Babon, Julianna Blagih, Alvin Pratama, Jaime Martin, Naomi Hawley, Jean Y Cappello, Pablo F Nieto, Julia I Ellyard, Nadia J Kershaw, Rebecca A Sweet, Christopher C Goodnow, Russell G Jones, Mark A Febbraio, Carola G Vinuesa, Vicki Athanasopoulos writes that T follicular helper cells (Tfh) are critical for the longevity and quality of antibody-mediated protection against infection. Yet few signaling pathways have been identified to be unique solely to Tfh development. ROQUIN is a post-transcriptional repressor of T cells, acting through its ROQ domain to destabilize mRNA targets important for Th1, Th17, and Tfh biology. Here, we report that ROQUIN has a paradoxical function on Tfh differentiation mediated by its RING domain: mice with a T cell-specific deletion of the ROQUIN RING domain have unchanged Th1, Th2, Th17, and Tregs during a T-dependent response but show a profoundly defective antigen-specific Tfh compartment. ROQUIN RING signaling directly antagonized the catalytic α1 subunit of adenosine monophosphate-activated protein kinase (AMPK), a central stress-responsive regulator of cellular metabolism and mTOR signaling, which is known to facilitate T-dependent humoral immunity. We therefore unexpectedly uncover a ROQUIN–AMPK metabolic signaling nexus essential for selectively promoting Tfh responses.

eLife digest

The immune system protects the body from invading microbes like bacteria and viruses. Upon recognizing the presence of these microbes, cells in the immune system are activated to destroy the foreign threat and clear it from the body.

A type of immune cell called T follicular helper cells (or Tfh for short) are formed during an infection and are essential for coordinating other immune cells to produce high-quality antibody proteins that attack the microbes. Without Tfh cells, life-long production of these protective antibodies is severely crippled, which can cause common variable immune deficiency and other serious immunodeficiency diseases. On the other hand, the body must also avoid generating excessive numbers of Tfh cells, which can lead to the production of antibodies that attack healthy cells of the body.

ROQUIN is a protein that inhibits the formation of Tfh cells and other types of active T cells. A region on the protein called the ROQ domain destabilizes particular molecules of ribonucleic acid (RNA) that are required for these specialist T cells to form and work properly. ROQUIN belongs to a large family of enzymes that have a so-called RING domain, which is a feature that enables these enzymes to attach tags onto specific target proteins to modify their activity or stability. However, it was not known whether the RING domain of ROQUIN was active.

Ramiscal et al. now address this question in mice. Unexpectedly, the experiments show that the RING domain is required to promote the formation of Tfh cells, but not other types of active T cells. This domain allows ROQUIN to repress an enzyme called AMPK, which normally blocks cell growth by regulating cell metabolism. The findings suggest that the different roles of the ROQ and RING domains allow ROQUIN to fine-tune the numbers of Tfh cells so that they remain within a safe range. In the future, these findings may aid the development of vaccines that are more efficient at generating protective Tfh cells to prevent infectious diseases.

Introduction

High-affinity and long-lasting humoral immunity against infection requires controlled cross-talk between limiting CD4+CXCR5highPD1highBCL6high T follicular helper (Tfh) cells and immunoglobulin-maturing germinal center (GC) B cells in secondary lymphoid tissues (King et al., 2008; Victora and Nussenzweig, 2012; Nutt and Tarlinton, 2011; Ramiscal and Vinuesa, 2013). As the GC largely consists of clonally diverse B cells, Tfh cells especially in narrow numbers are best at maintaining a selective pressure for B cell competition, favoring the survival of greater affinity antigen-responsive GC B cell clones (Pratama and Vinuesa, 2014; Victora and Mesin, 2014). Deregulation of Tfh cells can lead to faulty GC selection that may also seed the production of autoantibodies (Weinstein et al., 2012; Vinuesa et al., 2005; Kim et al., 2015; Linterman et al., 2009) and GC-derived malignancies such as follicular lymphoma (Rawal et al., 2013; Klein and Dalla-Favera, 2008). To date, the signals that exclusively govern Tfh cell differentiation over other T cell effector subsets remains poorly characterized.

ROQUIN (also called ROQUIN1; encoded by Rc3h1) acts to post-transcriptionally repress Tfh cells by binding effector T cell transcripts via its winged-helix ROQ domain (Schuetz et al., 2014; Tan et al., 2014; Schlundt et al., 2014) and recruiting proteins of the RNA decapping and deadenylation machinery (Athanasopoulos et al., 2010; Glasmacher et al., 2010; Leppek et al., 2013; Pratama et al., 2013; Yu et al., 2007; Vogel et al., 2013) as well as the endoribonuclease REGNASE-1 (Jeltsch et al., 2014). Some of its RNA targets include the Tfh-polarising Icos (Glasmacher et al., 2010) and Il6 mRNA (Jeltsch et al., 2014) as well as Ox40 (Vogel et al., 2013) and Tnf (Pratama et al., 2013) transcripts. In sanroque mice, an Rc3h1 missense point mutation, encoding for a Met199 to Arg substitution translates into a minor conformational shift in the RNA-binding ROQ domain (Srivastava et al., 2015) of ROQUIN and a loss of function in post-transcriptional repression. This leads to excessive Tfh growth and systemic autoimmunity (Linterman et al., 2009; Vinuesa et al., 2005). Complete ablation of ROQUIN results in unexplained perinatal lethality in C57BL/6 mice and selective deletion of ROQUIN in T cells does not lead to Tfh cell accumulation nor autoimmunity (Bertossi et al., 2011). The latter is at least in part explained by the existence of the closely related family member ROQUIN2 (encoded by Rc3h2), which has overlapping functions with ROQUIN (Pratama et al., 2013; Vogel et al., 2013). The ROQUINM199R mutant protein has been proposed to act as a ‘niche-filling’ variant that has lost its RNA-regulating activity (Pratama et al., 2013) but can still localize to mRNA-regulating cytoplasmic granules to prevent the compensatory activity of ROQUIN2.

ROQUIN contains a conserved amino terminal RING finger with two conforming zinc-chelating sites (Srivastava et al., 2015), despite an atypical aspartate as its eighth zinc ligand synonymous to RBX1 (Kamura et al., 1999). This suggests ROQUIN may function as an E3 ubiquitin ligase (Deshaies and Joazeiro, 2009) but, to date, no such enzymatic activity of the ROQUIN RING domain has been demonstrated in mammals. In vivo attempts to delineate the cellular pathways regulated by ROQUIN are made challenging due to the existence of multiple protein domains in the protein (Figure 1—figure supplement 1a). The Caenorhabditis elegans ROQUIN ortholog, RLE-1, acts through its RING domain to ubiquitinate DAF-16, a pro-longevity forkhead box O (FOXO) transcription factor homolog (Li et al., 2007). We did not find any evidence for molecular binding between ROQUIN and the fruitfly or mammalian FOXO orthologs (Drosophila melanogaster FOXO and Mus musculus FOXO1 or FOXO3a; data not shown) and therefore set out to understand the role of ROQUIN RING signaling in CD4+ T cell development and function by generating mice that selectively lack the ROQUIN RING zinc finger.

We previously demonstrated that ROQUIN RING-deleted T cells in mice 6 days after sheep red blood cell (SRBC) immunization can form normal early Tfh cell responses but fail to promote optimal GC B cell reactions (Pratama et al., 2013). Here, in mice that have developed robust Tfh-dependent GC responses toward SRBC or infected with lymphocytic choriomeningitis virus (LCMV), we identify a novel and unexpected role of the ROQUIN RING domain in selectively promoting mature antigen-specific Tfh cell responses while leaving unaffected the development of other CD4+ effector T cell lineages. ROQUIN directly binds to and limits adenosine monophosphate-activated protein kinase (AMPK), a tumor suppressor and central regulator of T cell glucose uptake and glycolysis (MacIver et al., 2011). Our data indicate that loss of AMPK repression by deletion of the ROQUIN RING domain promotes stress granule persistence. This in turn cripples mTOR activity, otherwise known to play a critical role in driving CD4+ effector T cell expansion (Delgoffe et al., 2009; 2011) and T-dependent antibody responses (Keating et al., 2013; Zhang et al., 2011; Gigoux et al., 2014; De Bruyne et al., 2015).

Results

The ROQUIN RING domain selectively controls Tfh cell formation

To examine the function of the ROQUIN RING domain in vivo, we generated two strains of C57BL/6 mice carrying either a germline deletion (designated ringless; ‘rin’ allele) or a T cell conditional deletion (Tringless; ‘Trin’ allele) of exon 2 in the Rc3h1 gene, which encodes the translation START codon and RING finger domain of the ROQUIN protein (Figure 1—figure supplement 1b, c and Pratama et al., 2013). In these mice, skipping of exon 2 resulted in splicing of exon 1 to exon 3 yielding an alternative in-frame Kozak translation initiation site at Met133 (Figure 1—figure supplement 1d, e). This predicted ROQUIN133-1130 protein product specifically lacks the RING domain (Figure 1—figure supplement 1f). Mice homozygous for the rin allele were perinatally lethal (Figure 1—figure supplement 1g–i), precluding T cell studies in intact animals. In contrast, Tringless mice were viable and showed no severe variations in thymic development and output of CD4 single positive T cells (Figure 1—figure supplement 2a–e). There were also no major changes in Th1 cell differentiation in Tringless mice infected with LCMV (Figure 1a), which predominantly yields LY6Chigh Th1 and LY6Clow Tfh virus-specific effector cells (Hale et al., 2013; Marshall et al., 2011). In Tringless animals immunized with SRBCs, the formation of Th1, Th2, Th17, and regulatory T cells also remained largely unperturbed (Figure 1—figure supplement 2f, g). This was mirrored in vitro with Tringless CD4+ naive T cells activated under Th1, Th2, Th17, or induced Treg (iTreg) polarizing conditions (Figure 1—figure supplement 2h) displaying maximal expression of intracellular TBET, GATA3, RORγT, and FOXP3 comparable to floxed wild-type T cell cultures (Figure 1—figure supplement 2i). Surprisingly in Tringless mice, there was an overall defective Tfh cell primary response to LCMV infection (Figure 1b–d) and to SBRC immunization (Figure 1—figure supplement 3a). ROQUIN RING-deficient T cells were also inefficient in supporting GC formation (Figure 1e, f and Figure 1—figure supplement 3b), which was associated with reduced IL-21 production (Figure 2a), a Tfh signature cytokine vital in supporting GC reactions (Liu and King, 2013).

By stimulating splenocytes ex vivo with GP61-80 peptide to identify virus-responsive IFNγ-producing Th1 cells (Figure 2b) and by examining splenic LYC6high Th1 cells amongst GP66-77+ tetramer stained T cells (Figure 2c), we verified that ROQUIN RING loss did not disrupt protective Th1 responses but caused a severe abrogation of virus-specific Tfh cells during LCMV infection (Figure 2d–f). Virus-specific T cells also showed significantly reduced expression of BCL6 (Figure 2g), an indispensible nuclear factor for Tfh cell terminal differentiation (Liu et al., 2013). Furthermore, we found an increased frequency of FOXP3+ T follicular regulatory (Tfr) cells within the total Tfh pool (Figure 2h) despite these Tfr cells not expressing a GP66-77 virus-specific T cell antigen receptor (TCR; Figure 2i). Nonetheless, as Tfr cells are negative regulators of GC reactions (Ramiscal and Vinuesa, 2013), their abundance may indicate augmented suppression of Tfh cells and long-term B cell responses.

ROQUIN undergoes RING-dependent autoubiquitination and directly limits AMPK activity

We next sought to determine the molecular basis for the ROQUIN RING domain as a determinant in protective Tfh cell responses. Several lines of evidence implicated an involvement of ROQUIN in the negative regulation of AMPK signaling: Rc3h1ringless fetuses displayed skeletal muscle atrophy of the thoracic diaphragm (Figure 1—figure supplement 1j), which is a characteristic phenotype of mice with overactive AMPK (Sanchez et al., 2012) and pointed to perinatal respiratory failure as the cause of the lethality. Also, AMPK over-expression in nematode worms has been shown to extend lifespan (Mair et al., 2011), an observation consistent with the phenotype of worms lacking the ROQUIN ortholog RLE-1 (Li et al., 2007). Since the AMPKα1 catalytic subunit is expressed in T cells and responds to TCR activation (Tamas et al., 2006), we tested the possibility of ROQUIN directly binding to this subunit of AMPK (encoded by Prkaa1). Upon ectopic expression in HEK293T cells, ROQUIN colocalized with AMPKα1 diffusely or in fine cytoplasmic speckles in resting cells and within larger cytoplasmic granules upon induction of oxidative stress (Figure 3a). We also observed colocalization of endogenous AMPKα1 within ROQUIN+ cytoplasmic granules in arsenite-treated primary C57BL/6 mouse embryonic fibroblasts (MEFs) (Figure 3b) with the use of an AMPKα1-specific antibody displaying no cross-reactivity toward the AMPKα2 subunit when ectopically expressed in HEK293T cells (Figure 3—figure supplement 1a). Unlike the AMPKα1 subunit, ectopically expressed AMPK β and γ regulatory subunits did not associate with ROQUIN+ cytoplasmic granules, although AMPKγ2 and AMPKγ3 exhibited generally diffuse cytoplasmic distribution (Figure 3—figure supplement 1b). We next determined if ROQUIN and AMPKα1 interacted by conducting in situ proximity ligation assays (PLAs) on primary C57BL/6 MEFs. Compared to control PLAs accounting for false interactions between endogenous AMPKα1 and non-expressed green fluorescent protein (GFP) detected by optimized anti-GFP immunostaining (Figure 3—figure supplement 1c), we found that endogenously expressed ROQUIN and AMPKα1 proteins localized with very close molecular proximity in both resting and arsenite-stressed cells (Figure 3c, d) at a frequency 15-fold higher or more than weak PLA interactions previously observed between ROQUIN and AGO2 (Srivastava et al., 2015). Moreover, we were able to coimmunoprecipitate ROQUIN and AMPKα1 when over-expressed in HEK293T cells (Figure 3—figure supplement 1d) or expressed endogenously in the mouse T lymphoblast line EL4 cells (Figure 3e). Together with the PLAs, this indicated that ROQUIN bound specifically with the α1 subunit of AMPK and that under physiological conditions, the two proteins could form a stable complex.

( Author are from Australian National University, Australia; Baker IDI Heart and Diabetes Institute, Australia; Walter and Eliza Hall Institute of Medical Research, Australia; McGill University, Canada; Garvan Institute of Medical Research, Australia)

The Article can be read in full in eLife  DOI 

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First Published: September 24: 2015