Abstract
APEX is an engineered peroxidase that catalyzes the oxidation of a wide range of substrates, facilitating its use in a variety of applications, from subcellular staining for electron microscopy to proximity biotinylation for spatial proteomics and transcriptomics. To further advance the capabilities of APEX, we used directed evolution to engineer a split APEX tool (sAPEX). Twenty rounds of FACS-based selections from yeast-displayed fragment libraries, using three different yeast display configurations, produced a 200-amino acid N-terminal fragment (with 9 mutations relative to APEX2) called “AP” and a 50-amino acid C-terminal fragment called “EX”. AP and EX fragments were each inactive on their own but reconstituted to give peroxidase activity when driven together by a molecular interaction. We demonstrate sAPEX reconstitution in the mammalian cytosol, on engineered RNA motifs within telomerase noncoding RNA, and at mitochondria-endoplasmic reticulum contact sites.
Introduction
APEX2 is an engineered variant of soybean ascorbate peroxidase that, unlike the commonly used horseradish peroxidase (HRP), retains activity when expressed in the cytosol, mitochondria, and other reducing environments within the cell1,2. This feature of APEX2, in addition to its versatile ability to catalyze the H2O2-dependent one-electron oxidation of a wide variety of small molecule substrates, has led to its widespread use for a variety of applications, including proteomic mapping of organelles2–6, proximity labeling of protein interactomes7–9, spatial mapping of cellular RNA10, electron microscopy1,11–16, H2O2 sensing17, and protein topology determination1,2,16.
APEX2 is typically fused to a protein or peptide to target it to a subcellular region or macromolecular complex of interest. For instance, we have targeted APEX2 to the outer mitochondrial membrane (OMM) and the endoplasmic reticulum membrane (ERM) of mammalian cells by fusing the APEX2 gene to transmembrane domains of proteins native to these subcellular locations4,16. These constructs were used for both EM16 and proteomic analysis4 of the OMM and ERM. While this APEX2 fusion strategy has enabled the study of many cellular regions and organelles, there are numerous compartments and structures to which APEX cannot be selectively targeted. For example, there is great interest in the biology of organelle-organelle contact sites, such as the junctions between mitochondria and ER, which participate in calcium signaling18,19, lipid synthesis20–23, and mitochondrial fission24,25. Yet all candidate protein fusions that could potentially target APEX2 to these contact sites, such as to the proteins Drp124, Mfn226–28, SYNJ2BP14, and PDZD829, would also target the peroxidase to locations outside of mito-ER contacts, such as throughout the cytosol30, along the cytoskeleton31, or over the entire OMM4.
Another application for which the APEX2 genetic fusion strategy may be unsuitable is profiling the interactomes of specific cellular RNAs. While several robust methods can identify RNAs that interact with specific proteins of interest32–34, the converse problem—identifying proteins that interact with a particular RNAs—is much more challenging using existing methods. One could envision fusing APEX2 to a high-affinity RNA-binding protein (RBP; for example, the bacteriophage MS2 coat protein35), allowing the peroxidase to be ectopically targeted to transcripts that are tagged with that RBP’s cognate RNA motif. However, a major concern would be the excess pool of catalytically active APEX2-RBP fusion protein that is not docked to the tagged RNA and can therefore produce off-target labeling that masks the specific signal.
A general solution to both these, and related, problems could be a split form of APEX2, in which two inactive fragments of APEX2 reconstitute to give an active peroxidase only when they are physically co-localized (Figure 1A). One could use an intersectional approach to restrict APEX2 activity specifically to sites of interest–such as mito-ER contacts, or specific RNA binding sites – thus eliminating the background labeling from off-target peroxidase activity.
Although split protein reporters have been developed from green fluorescent protein36,37, HRP38, dihydrofolate reductase39, ubiquitin40, luciferase41–43, beta-galactosidase44–47, TEV protease48, Cas949–52, and BioID53, splitting APEX2 presents new challenges. First, APEX2 requires a heme cofactor for its activity, and many cut sites would split the heme-binding pocket between the two fragments (Figure 1B). Second, in order for split APEX2 to be useful for a broad range of applications, the inactive fragments should have relatively low affinity for one another, such that reconstitution only occurs when the fragments are driven together by a molecular interaction. Not many known split proteins have low-affinity fragments, and it is challenging to engineer such a property in conjunction with high activity upon reconstitution.
To address the need for an interaction-dependent proximity labeling tool, we describe here the development of an evolved split APEX2 (sAPEX) system. Using a novel yeast display-based directed evolution approach that incorporates positive and negative selection steps, we have attempted to minimize interaction-independent association of the peptide fragments while maintaining high peroxidase activity upon reconstitution. Our resulting sAPEX fragment pair diverges substantially from its parental sequence and shows interaction-dependent reconstitution in multiple contexts in living mammalian cells. Our sAPEX tool adds to the proximity labeling toolkit, and in the future, it should extend the utility of APEX-based approaches to new areas of biology at higher spatiotemporal resolution.
Results
Structure-guided screening of potential APEX2 cut sites
We first sought to identify promising cut sites in the APEX2 enzyme using a chemically-inducible protein association system as a test platform. We selected 24 potential cut sites within solvent-exposed loops and turns between secondary structural elements (alpha helices and beta sheets) based on the crystal structure of wild-type ascorbate peroxidase54 (Figure 1B). Cut sites were also selected to avoid the creation of hydrophobic termini that might destabilize the fragments. All fragments were expected to lack a complete heme-binding pocket, with the exception of the large fragments produced from cut sites 7/8 and 29/30. We cloned each of the 24 fragment pairs as fusions to FKBP and FRB, whose interaction can be induced with the small molecule rapamycin (Figure 1C). We introduced the fragment pairs into HEK 293T mammalian cells using transient transfection and evaluated peroxidase activity in the presence or absence of rapamycin. Catalytic activity was detected using an established assay based on the membrane-permeable fluorogenic probe Amplex UltraRed, which is colorless but is converted into the red fluorophore resorufin upon peroxidase-catalyzed oxidization55,56.
Of the 24 fragment pairs tested, seven produced significant resorufin product, indicative of reconstituted activity (Figure 1D). For all cut sites except 7/8, much stronger activity was detected in the presence of rapamycin compared to when rapamycin was omitted, indicating that reconstitution was dependent on a protein-protein interaction. In the case of cut site 7/8, resorufin fluorescence was observed not only when rapamycin was omitted, but also when the large C-terminal fragment (amino acids 8-250) was expressed on its own (Figure S1), making this cut site unsuitable for our purposes. Cut sites within the heme binding cavity (130/131, 134/135, 146/147, 170/171, 173/174, and 181/182) produced fragments that completely failed to reconstitute activity (see Figure 1B and Figure 2 for recolored full length APEX2 (PDB:10AG)).
To more finely map the optimal cut sites, we performed a second round of screens, evaluating cut sites 1–3 residues away from promising sites identified in our initial screen (Figures 1E and S1). We ultimately identified three optimal cut sites—51/52, 89/90, and 200/201—all of which fell in solvent-exposed loops distal (>15 angstroms away) from the APEX2 active site and heme-binding pocket. For each cut site pair, controls in which either the N- or C-terminal fragments were expressed alone lacked any detectable peroxidase activity (Figure S1).
To assess the efficacy of our split APEX2 fragment pairs for applications in EM and proximity biotinylation, we evaluated their activity using the small molecule substrates diaminobenzidine (DAB)1,16 and biotin phenol (BP)2–4, respectively. Despite positive results in the highly sensitive Amplex UltraRed assay (Figure 1D-E), all three split APEX2 pairs produced far weaker DAB and BP staining than full-length APEX2 (Figure 3B-D and data not shown). For example, HEK 293T cells expressing full-length APEX2 exhibited very strong DAB staining after 15 minutes, while split APEX2 (cut site 200/201)-generated DAB stain under identical conditions was hardly detectable (Figure 3B). We found that the recently reported57 neighboring cut site, 201/202, exhibited activity similar to 200/201 (Figure S2).
A yeast display-based platform for split APEX2 evolution
The low activity of split APEX2 could be caused by a variety of factors, including poor stability, misfolding, aggregation of the fragments, or distorted geometry of the reconstituted active site and heme binding pocket, which could lead to low catalytic efficiency and/or poor heme recruitment. It is difficult to assess each of these potential problems, and even more difficult to fix them in a rational manner. Hence, we turned to directed evolution, which we and others have harnessed to improve or alter the properties of enzymes16,38,58–62. We selected yeast display-based directed evolution (Figure 2A) because, in contrast to other strategies such as high-throughput screening and phage display, yeast display allows processing of large mutant libraries (>107) with large dynamic range – enrichment based on Fluorescence-Activated Cell Sorting (FACS) enables separation of highly active catalysts from moderately active ones, not just from inactive catalysts.
Our initial yeast display selection scheme exploited the yeast mating proteins Aga1p and Aga2p, which are displayed on the yeast cell surface and are joined by two disulfide bridges (Figures 2A and S3A). A library of yeast cells was generated, with each cell displaying on its surface a different mutant of the N-terminal split APEX2 fragment via fusion to the mating protein Aga2p. All cells concomitantly displayed the same C-terminal split APEX2 fragment as a fusion to Aga1p. The fragments were allowed to reconstitute for ~20 hours post-induction of protein expression. To read out the resulting peroxidase activity, APEX2-mediated biotinylation was initiated with biotin-phenol and H2O2, using standard conditions16. Yeast cells that display active reconstituted peroxidase under these conditions should promiscuously biotinylate proteins on their cell surface, which can be quantified using FACS (Figures 2A and S3A).
To establish this selection platform, we first created yeast Aga1p/Aga2p fusion constructs using the three promising split APEX2 pairs identified in the above screen. Because the 51/52 and 89/90 fragment pairs expressed poorly in yeast and gave no detectable activity on the yeast surface (data not shown), we proceeded with directed evolution of the 200/201 fragment pair. The C-terminal fragment (amino acids 201–250 of APEX2; henceforth called “EX”) was held constant while the N-terminal fragment (amino acids 1–200 of APEX2, called “AP-0”) was mutagenized using error prone PCR. Sequencing showed that our AP-0 library contained an average of 1.4 amino acid mutations per clone (Methods).
We performed four rounds of selection and observed that the activity of the yeast pool (measured by FACS) progressively increased (Figure S3B). We isolated 12 individual clones, characterized their activity on yeast using FACS and in the mammalian cytosol using microscopy, and combined mutations that appeared to be beneficial. The result was “AP-1”, which contains 3 mutations relative to the original APEX2. In a side-by-side comparison to the original split APEX2 fragment pair (AP-0 + EX) in HEK 293T cells, AP-1 shows noticeably improved activity in both DAB and BP labeling assays (Figure 3B).
To further improve the reconstituted activity of split APEX2, we performed another set of yeast selections in which the C-terminal EX fragment was not co-displayed on Aga1p but instead added as a purified, soluble protein (Figure S4A). This configuration allowed us to precisely control the concentration of EX added and the time of incubation, and to select for mutations that improved AP-1 expression and stability in the absence of EX co-translation. However, because the fragments only encountered each other on the yeast surface, where endogenous heme is not available, it was necessary to supply exogenous heme to the cells both during and prior to EX fragment addition. Four rounds of selection using this scheme produced the clone “AP-2”, which has three mutations beyond those in AP-1.
In a third-generation effort, to drive the fragments together using a protein-protein interaction (PPI) we again added EX as a soluble protein, but we used an artificially designed coiled-coil system, ACID-p1 and BASE-p163, to recruit EX to the proximity of the N-terminal fragment (AP-2). This configuration mimics the split APEX2 reconstitution that would occur in our target biological applications. Four rounds of selection with gradual reduction of EX concentration and incubation time produced clone “AP-3”, which incorporates one additional mutation relative to “AP-2” and had noticeably higher activity than preceding clones in both DAB and BP labeling assays in HEK 293T cells (Figure 3B-C and Figure S5).
FACS-based negative selections for reduced fragment affinity
An ideal split APEX2 fragment pair would have high catalytic activity when reconstituted, but low intrinsic binding affinity between the fragments, such that the fragments would only reconstitute when driven together by a PPI (Figure 1A). The clone AP-3, obtained after three generations of directed evolution, has much greater reconstituted activity (with EX) than does the original template (AP-0), but this activity was not dependent on PPI–induced co-proximation: using FRB and FKBP fusion proteins in HEK 293T cells, for example, we observed considerable DAB and BP signal in the absence of rapamycin (Figure 3B-C).
To preserve high reconstitution activity but decrease fragment affinity, we devised a new yeast display selection scheme that alternates between positive and negative selection rounds (Figure 2A). For the positive selection, we supplied the purified EX protein fused to the BASE-p1 helix that facilitates recruitment to Aga1p-ACID-p1. We performed BP labeling followed by streptavidin-phycoerythrin and anti-myc antibody staining. FACS was used to enrich cells with high SA/myc intensity ratios, as above. For the negative selection, we incubated the yeast with EX protein lacking BASE-p1 coil for extended periods of time (3 to 25 hours). AP-3 mutants that reconstituted with EX under these PPI-independent conditions were eliminated by FACS.
Starting with a library of 4.8 × 108 AP-3 variants, we performed two rounds of positive selection and six rounds of negative selection (Figure S6A). By round 8, the yeast population was substantially depleted of cells that could reconstitute APEX2 activity upon addition of EX lacking the BASE-p1 coil (Figure S6B). We isolated 4 unique clones, characterized them by FACS, combined beneficial mutations, and re-tested in the mammalian cytosol. These experiments resulted in AP, the final split APEX2 (sAPEX) N-terminal fragment, which contains 9 mutations compared to the original APEX2 sequence. Mapping the positions of these 9 mutations onto the structure of wild-type ascorbate peroxidase, we observe that all lie in solvent-exposed regions, and none are at the interface between AP and EX (Figure 2D). Interestingly, half of the mutations are adjacent to cut sites we screened in Figure 1D-E, and many of the mutations were clustered in specific regions of the protein structure (Figure 2D).
Characterization of enriched clones
Having completed 20 rounds of selection using three different yeast display configurations, we characterized key clones side-by-side. First, we prepared yeast displaying AP-0 (the starting template), AP (N-terminal fragment of final sAPEX), full-length APEX2, and AP-1 to AP-3 mutants, as fusions to Aga2p. Aga1p on these cells contained the ACID-p1 coil. We then supplied EX protein, either fused to (Figure 2A, top row) or lacking (bottom row) the BASE-p1 coil for proximity-dependent reconstitution. Figure 2B shows that the streptavidin-phycoerythrin (PE) staining (indicating reconstituted peroxidase activity) progressively increases from the template AP-0 to the finalized AP. However, the signal in the bottom row, reflecting proximity-independent reconstitution with EX (lacking the BASE-p1 coil), also increases, which is undesirable. After the implementation of negative selections in Generation 4, however, the untagged EX signal decreases for the AP clone. The EX+BASE-p1 coil signal remains high for AP, although not quite as high as that seen for AP-3. These observations on yeast indicate that the selections worked as desired and that our final clone AP combines the features of high reconstitution activity with low proximity-independent reconstitution.
We next tested whether these trends would hold in the mammalian cell cytosol. This environment is quite different from the yeast cell surface, as it is 37 °C instead of 30 °C, and a reducing rather than oxidizing environment. We also wished to test the sAPEX clones as soluble proteins rather than as membrane-anchored constructs with restricted geometry. Hence, we expressed the N- and C-terminal fragments from each stage of directed evolution as fusions to FKBP and FRB, respectively (Figure 3A). We first compared peroxidase activity with or without rapamycin using our DAB assay (relevant for EM applications1,16), which is much less sensitive than both Amplex UltraRed and BP assays, thus enabling us to rigorously compare the activity of our sAPEX fragment pairs (Figure 3B). The original sAPEX template, AP-0, gave barely detectable DAB staining, while AP-1 was dramatically improved, and AP-3 gave the strongest staining. However, as observed in yeast, AP-3 also gave significant signal in the absence of rapamycin, indicating high PPI-independent reconstitution. In contrast, the final AP + EX clones displayed high DAB staining in the presence of rapamycin, but nearly undetectable staining in the absence of rapamycin.
Analogous experiments using the BP assay—relevant for spatial proteomics2,3 and transcriptomics64—showed a similar trend (Figure 3C and Figure S7). While AP-3 showed high activity following rapamycin treatment, it also revealed background labeling in the – rapamycin condition. In contrast, the final sAPEX pair, AP + EX, displayed +rapamycin activity comparable to that of AP-3, but minimal – rapamycin activity. We also characterized BP labeling by lysing the cells and blotting the cell lysate with streptavidin-HRP (Figure 3D). The PPI-dependence of sAPEX makes it a versatile tool for cellular applications such as those outlined in the introduction.
sAPEX reconstitution on a target RNA
As APEX-catalyzed proximity biotinylation has demonstrated utility for mapping protein interactomes7–9, there has been interest in using APEX2 to also map the interactomes of target nucleic acids – RNAs and individual genomic loci. In theory, this could be accomplished by fusing full-length APEX2 to programmable RNA- or DNA-binding proteins (such as CRISPR-Cas13d or Cas9, respectively)65–67 that specifically target the RNA or genomic locus under investigation. Alternatively, target RNAs or DNA loci could themselves be tagged with motifs that specifically bind APEX2 fusion proteins68. However, each of these approaches would be plagued by pools of excess catalytically active APEX2 that is unbound to the target of interest. This is exemplified by recent studies that used dCas9 to recruit APEX2 to specific genomic loci66,67, and a separate study that used the bacteriophage MS2 RNA coat protein (MCP) to recruit promiscuous biotin ligase variants to RNAs appended with the cognate MS2 RNA motif68. An analogous APEX2-MCP fusion system would also likely suffer from background biotinylation that overwhelms the specific signal from RNA-bound APEX-MCP.
We reasoned that our sAPEX system could potentially be used to alleviate this problem by fusing the evolved AP and EX fragments to orthogonal RNA-binding proteins, and selectively reconstituting APEX2 peroxidase activity on target RNAs that are appended with both of the cognate protein-binding RNA motifs (Figure 4A-B). As proof-of-principle, we sought to apply this strategy to TERC, the noncoding RNA component of the telomerase ribonucleoprotein (RNP), which synthesizes the ends of chromosomes in many clades of eukaryotes69. In addition to providing the template for telomere synthesis, the TERC ncRNA is also thought to serve as a structural “scaffold” onto which the other holoenzyme components assemble70. Critically, proper biogenesis of functional telomerase RNPs can be recapitulated when TERC RNA is transiently expressed from a plasmid, even if the RNA is appended with exogenous sequences at its 5′ end71. We therefore designed a series of variants in which the TERC 5′ terminus is appended by cassettes of motifs recognized by RNA-binding proteins. For this purpose, we chose to express AP and EX as fusions to the MS2 and PP7 bacteriophage nucleocapsid proteins—respectively termed MCP and PCP—which recognize disparate RNA motifs with exquisitely high affinity and specificity (cognate RNA KD ~1nM, non-cognate RNA KD ~1mM)35,72,73,74 (Figure 4A-C). Anticipating that sAPEX reconstitution might be sensitive to the specific geometry with which the AP and EX fragments are co-proximated, we designed a series of TERC variants that positioned MS2 and PP7 stem loops in different orientations (Figure S8).
Expression of MCP–AP and PCP–EX resulted in biotinylation that was dependent on the presence of a TERC RNA bearing both of the cognate MS2 and PP7 motifs (Figure 4C and Figure S9). Critically, we observed no such biotinylation when individual MS2 and PP7 hairpins were localized on separate TERC RNA constructs. Moreover, the labeling pattern appeared punctate, as would be predicted from localizing peroxidase activity to the discrete subnuclear foci characteristic of interphase telomerase75. Our data suggest that sAPEX activity can be specifically reconstituted on a target RNA by nucleating protein fragment assembly on a structured RNA cassette.
sAPEX reconstitution at mitochondria-ER contacts
In mammalian cells, an estimated 5–20% of the mitochondrial outer membrane makes intimate contact (<70 nm gap) with the membrane of the endoplasmic reticulum (ER)19. These mito-ER contacts are thought to be important for a variety of functions and signaling processes, from mitochondrial fission to lipid synthesis18,19,20–23,24,25. Recent work has identified proteins that reside at mito-ER contacts and may play a role in tethering the membranes together4,29. A major goal of this field is to comprehensively characterize the molecular composition of these contact sites to better understand how they mediate important cellular processes. To pave the way for such future efforts, we tested whether sAPEX activity could be reconstituted at mito-ER contact sites.
Mito-ER contacts are delicate and easily perturbed structures. Overexpression of various proteins, such as SYNJ2BP or even green fluorescent protein, can lead to dramatic distortion of one or both organellar membranes4,76. Because our optimized sAPEX fragment pair, AP + EX, does not exhibit PPI-independent reconstitution, we reasoned the tool may be suitable for reconstitution at mito-ER contacts without major perturbation of organellar structure. Figures 4D-F and S10 show AP-FKBP targeted to the OMM and EX-FRB targeted to the ERM in COS7 cells, which are flat and thin, facilitating visualization of mitochondria and ER structures. We observed BP/streptavidin staining in cells treated with rapamycin for 30 minutes. Consistent with the PPI-dependence of sAPEX reconstitution, no BP labeling was observed when rapamycin was omitted. We also stained the COS7 cells for mitochondrial and ER markers and observed minimal disruption of organellar morphology, both before and after rapamycin addition (Figure S10B-C).
To examine sAPEX at mito-ER contacts at higher resolution, we examined mito-ER reconstituted sAPEX activity using electron microscopy (EM). We fixed the above COS7 cells, and overlaid with DAB and H2O2 to allow reconstituted sAPEX to catalyze the oxidative polymerization and local deposition of DAB1. Bright field imaging showed dark threads corresponding to stained mitochondria in the 30-minute rapamycin-treated samples, but not in the untreated samples (Figure 4G). We next stained the rapamycin-treated samples with OsO4 to deposit electron-dense osmium on the DAB polymer, then embedded and sectioned the samples for EM. EM imaging revealed a dark stain, corresponding to regions of reconstituted sAPEX activity, exclusively at contact sites between mitochondria and ER (Figure 4H). Two zoomed views show that DAB staining filled the ~25 nm gap between the OMM and ERM, but was absent from isolated ER and mitochondrial membranes. Notably, the mitochondrial and ER membranes were not grossly perturbed, as was observed in previous studies in which overexpressed reporters induced non-native mito-ER contacts and organelle aggregation16.
Discussion
Using a combination of rational design and yeast-display directed evolution, we have engineered a low-affinity sAPEX protein complementation assay (PCA) with robust activity upon reconstitution driven by co-proximation. After three generations of evolution, we evolved a variant of split APEX2 with robust reconstitution, but which was prone to PPI-independent reconstitution. To overcome this limitation, our final directed evolution strategy implemented a negative selection that eliminated yeast clones with high fragment affinity. The final engineered sAPEX fragments, AP and EX, possess a total of nine mutations relative to the starting APEX2 template, clustered at solvent exposed regions, that collectively improve the PPI dependence of this system while maintaining high catalytic activity of the reconstituted form. This yeast-display platform could be extended to engineer other PCA systems. High-affinity fragment pairs such as those of split GFP result in spontaneous and irreversible reconstitution77; similarly, split HRP irreversibly reconstitutes in the ER without a PPI38. Split YFP utilizes fragments that reconstitute in a more PPI-dependent manner78,79, but it still suffers from background fluorescence, especially at high expression levels, and from irreversible YFP self-assembly80,81.
Given the purportedly broad array of novel RNAs and RNA-mediated processes that have eluded mechanistic dissection82 and the growing number of diseases now thought to be mediated by aberrant RNA-protein interactions83,84, there is a great need for identifying the interaction partners of specific RNAs. Conventional approaches address this problem by capturing and enriching a target RNA along with its bound interaction partners, either using affinity-tagged antisense oligonucleotides that isolate endogenous transcripts85–88, or by affinity tagging the transcript of interest itself89,90. However, these approaches are often confounded by the abundance of the target transcript, the unpredictable performance of RNA-based affinity tags91, and nonspecific RNA-protein interactions formed during lysis and enrichments92.
Using RNA scaffolding motifs to target the reconstitution of sAPEX eliminates high off-target background signal from protein overexpression. sAPEX demonstrates promise for BP labeling around specific RNAs, which indicates potential future applications to elucidate interactomes and interaction partners of an RNA of interest. Moreover, since the RNA domain we used to recruit AP and EX was designed to be functionally independent of the RNA to which it is appended, it should in theory be adaptable to other RNA targets93. A downside to this approach, however, is the need to express the transgenic, tagged RNAs that may not properly fold, localize, or function. As an alternative strategy, one might use RNA-targeting CRISPR-Cas systems to recruit AP and EX to endogenously expressed RNAs65. This approach may require substantial optimization to juxtapose the AP and EX fragments in the proper orientation, and may be influenced by the sequence, structure, or proteome of the targeted RNA.
The ability to reconstitute APEX2 activity at an organellar junction represents a promising step towards gaining greater understand of subcellular locales that were previously intractable to genetic targeting. We found that the reconstitution of sAPEX at mito-ER contact sites was PPI-dependent, despite multiple days of co-translation of the sAPEX fragments on the membranes of these organelles that are known to come into contact. Importantly, sAPEX did not greatly perturb the morphology of these organelles, as shown by fluorescence and electron microscopy. We note that some optimization was required to attain this PPI-dependence; for instance, we changed the promoter of OMM-FKBP-EX from CMV to human ubiquitin promoter (hUBC) to reduce the overall protein expression level, which reduced PPI-independent reconstitution. sAPEX potentially provides the ability to investigate and map the protein and RNA residents at other organelle-organelle contact sites beyond mito-ER junctions.
Unlike most other PCAs, sAPEX possesses the versatile capability to react with many different substrates, enabling its use for a wide array of applications. Because sAPEX activity can be reconstituted in highly specific subcellular regions that are intractable for single-gene constructs, it expands the toolkit of proximity labeling technologies.
Acknowledgements
FACS was performed at the Koch Institute Flow Cytometry Core (MIT) and Stanford Shared FACS Facility. S. Han (Stanford) synthesized neutravidin AlexaFluor647. This work was supported by NIH grants to A.Y.T. (R01-CA186568), to D.B. (R01-GM086197), and to M.H.E. (P41-GM103412) for support of the National Center for Microscopy and Imaging Research. D.M.S. was supported by HHMI/Jane Coffin Childs Fellowship and by J. Rinn (U. C. Boulder). T.C.B. was supported by Dow Graduate Research and Lester Wolfe Fellowships.
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