Isolation of Subcellular Ribosome Subpopulations Based on Recombinant Peptide Tag-Specific Location-Restricted Illumination-Enhanced Biotinylation

Summary

AviTag-specific Location-restricted Illumination-enhanced Biotinylation (ALIBi) is a novel optogenetically activated method developed for labeling and affinity-purifying subcellular populations of ribosomes for downstream sequencing and proteomic analysis.

Abstract

Growing evidence suggests that mRNA translation is a highly compartmentalized process within a cell, and that subcellular trafficking and localization of specific mRNAs is key to ensuring that proteins with compartment-specific functions are produced in the ideal milieu and in appropriate quantities. However, techniques for subcellular isolation and characterization of the ribosomes that translate these mRNAs have been limited to date, such that much remains unknown about how the composition of translational machinery contributes to regulation of localized mRNA translation.

Here, we demonstrate AviTag-specific Location-restricted Illumination-enhanced Biotinylation (ALIBi), a method that combines epitope tagging, a newly developed optogenetically activated split-biotin ligase, and affinity purification to rapidly and specifically label and isolate ribosomes localized to any subcellular compartment of interest. First, CRISPR/Cas9 editing is used to fuse an AviTag peptide, a tobacco etch virus (TEV) protease cleavage site, and a FLAG epitope tag to a ribosomal protein. The split biotin ligase, fused to an organelle-targeting domain, is expressed in this cell line and is inactive under normal biotin concentrations. Upon activation by supplemental biotin and blue light illumination, the ligase biotinylates AviTagged ribosomes in the immediate vicinity, allowing for affinity purification of biotinylated ribosomes and associated proteins and mRNAs on streptavidin-coated beads. Non-denaturing elution via TEV protease cleavage yields samples suitable for downstream characterization of core ribosomal proteins, ribosome-associated proteins, and ribosome-bound mRNAs via RNA sequencing or mass spectrometry proteomics.

In this protocol, we review design principles for fusing AviTag to a ribosomal protein and targeting the split biotin ligase enzyme to the organelle of interest. We demonstrate activation of the ALIBi system, cell lysis, affinity purification, and sample elution. Finally, we discuss typical results and troubleshooting.

Introduction

There is an important need for tools that can isolate biological samples from specific subcellular compartments to better investigate how subcellular organization and trafficking factor into cellular processes. mRNA translation is a key example of such a process: growing evidence suggests that translation is highly compartmentalized and, in particular, that many mRNAs are localized to the organelles where their encoded proteins will reside^1,2. However, less is known about the subcellular localization of translational machinery. Ribosomes begin their existence in the nucleolus and nucleus, where assembly from ribosomal proteins (RPs) and ribosomal RNAs (rRNAs) begins. They are then exported to the cytoplasm for the final stages of assembly after which they begin translating mRNAs^3,4. Canonical sites of translation include the cytoplasm and endoplasmic reticulum; the latter is where integral membrane proteins and proteins destined for membrane-bound organelles or extracellular secretion are synthesized⁵. More recently identified sites of localized translation include the mitochondria, peroxisome, and neuronal processes^{6,7,8,9,10,11,12}. Interestingly, evidence is also emerging that translational machinery features such as ribosome-associated proteins, post-translational modifications, rRNA modifications, and non-canonical stoichiometry of core ribosomal proteins are involved in regulatory mechanisms that govern translation of specific mRNAs and parameters such as translation fidelity and rate^{13,14,15,16,17,18,19,20}. For example, hundreds of proteins have been identified that interact with the ribosome and could potentially lead to differences in subcellular ribosome composition¹³. However, techniques for isolating subcellular ribosome populations have been limited to date; thus, much remains unknown about how the composition of translational machinery contributes to regulating localized mRNA translation. The advent of versatile methods for isolating subcellular ribosome populations, particularly those compatible with mammalian model systems, will allow for exploration of these translation machinery features at the subcellular level.

The earliest methods for isolating subcellular fractions containing ribosomes were based on principles of sequential lysis and centrifugation^21,22. Typically, these methods are limited to certain organelles and often require extensive processing times. A more recent method is based on the biotin ligase BirA, which specifically biotinylates an AviTag peptide substrate^10,23,24. The BirA can be genetically localized to the organelle of interest in cells where ribosomes have been tagged with AviTag. However, because BirA is active at physiologic concentrations of biotin, it is necessary to culture cells under conditions of biotin depletion in order to avoid constitutive and non-specific biotinylation of AviTag-bearing ribosomes^10,24. This limits the use of BirA in cell types that are sensitive to biotin depletion²⁵. Finally, promiscuous biotinylation methods have been developed that use engineered enzymes such as BioID, TurboID, and APEX^26,27,28. Upon induction, these enzymes produce a reactive biotin intermediate that covalently modifies any lysine residues within a radius of the enzyme. Promiscuous biotinylation methods are highly useful for mapping subcellular proteomes, but less informative for distinguishing proteins in the general vicinity of localized ribosomes as opposed to proteins that are in closer proximity with them.

Here, we demonstrate our recently developed method for labeling and isolating subcellular ribosome populations, called AviTag-specific Location-restricted Illumination-enhanced Biotinylation (ALIBi). This method leverages an engineered split BirA fused to optogenetic enhanced Magnet (eMag) switches (Figure 1A) that heterodimerize under blue light illumination²⁹; and a peptide tag consisting of FLAG epitope, tobacco etch virus (TEV) protease cleavage site, and AviTag (FTA) fused to the Rpl31 ribosomal protein (Figure 1B). The split enzyme can be genetically targeted to selected organelles, is inactive at physiologic concentrations of biotin, and can be reconstituted within minutes upon blue light illumination and the addition of supplemental biotin (Figure 1C-E). Subsequent cell lysis, affinity purification, and non-denaturing enzymatic elution via TEV protease cleavage yields ribosomes and co-purifying molecules such as mRNAs and ribosome-associated proteins, which are suitable for downstream analytic methods such as RNA sequencing and mass spectrometry proteomics (Figure 1A). Therefore, this method reveals not only ribosome composition but also the mRNAs to which ribosomes are bound. In a recent article describing the seminal application of ALIBi to characterize subcellular ribosome populations, we successfully targeted the split enzyme to the cytoplasm, nucleolus, nucleus, endoplasmic reticulum, plasma membrane, and mitochondria (Table 1)³⁰. The resulting comparison of translational machinery between these subcellular compartments led to novel and unexpected findings regarding translation at the endoplasmic reticulum and mitochondria, as well as potential leads for future investigation of ribosome biogenesis factors in the nucleus and nucleolus.

This protocol contains a detailed demonstration of transfection and activation of the ALIBi system, cell lysis, affinity purification, and sample elution. We also discuss how to verify successful affinity purification via western blot and make recommendations for performing downstream RNA sequencing or mass spectrometry (MS) proteomic analysis (see Supplemental File 1). This protocol is written for experiments using mouse embryonic stem cells (mESCs) expressing Rpl31 fused to FTA (L31-FTA) homozygously from the endogenous Rpl31 locus; this cell line is available from the authors upon request. The strategy for generating this cell line is described in Supplemental File 1 and in Supplemental Figure S1 and Supplemental Figure S2. Plasmids encoding the split enzymes targeted to several organelles are available on Addgene (catalog numbers #235608 to 235643). The design principles for targeting the split enzymes to these organelles is described in Supplemental File 1. The protocol assumes that each sample originates from mESCs grown in a 10 cm plate. We have found that the 10 cm scale yields sufficient material for mass spectrometry proteomics, while the 6-well scale is sufficient for western blots or RNA sequencing.

Protocol

1. mESC cell culture

NOTE: Prior to ALIBi activation and cell lysis, mESCs should be handled using standard sterile technique for cell culture. Centrifugation of intact mESCs should be performed at 200 × g for 3 min at room temperature unless otherwise specified. Prewarm media to 37 °C in a water bath before use unless otherwise specified. mESCs should be passaged into a fresh tissue culture plate that has been gelatinized overnight (see step 1.1).

1. The day prior to thawing L31-FTA mESCs, coat the bottom of a 10 cm plate with 5 mL of 0.1% gelatin. Incubate at 37 °C overnight.
2. Combine mESC media components (Table 2) and pass through a 0.2 µm filter. Store at 4 °C for up to 1 month.
3. On the day of thawing, warm a 5 mL aliquot of media for diluting the thawed cells and a 10 mL aliquot for plating them.
4. Retrieve a cryovial containing ~5 × 10⁶ L31-FTA mESCs from liquid nitrogen storage. Immediately thaw by swirling the vial bottom in a 37 °C water bath.
5. Gently mix 0.5–1 mL of media from the 5 mL aliquot with the cryovial contents as the vial capacity allows, then transfer the cryovial contents back to the 5 mL media aliquot.
6. Pellet the cells by centrifugation. Aspirate the supernatant.
7. Resuspend the cell pellet with the 10 mL media aliquot.
8. Aspirate the gelatin from the 10 cm plate and immediately transfer cell suspension into the plate. Incubate at 37 °C.
9. Every 24 h (except on passaging days), aspirate old media from the plate and replace with 10 mL of fresh media.
10. When colonies are 70%–80% confluent (typically 48 h after plating), aspirate the old media and rinse twice with 10 mL of Dulbecco’s phosphate-buffered saline (DPBS).
11. After aspirating the DPBS, coat the plate with 2 mL of 0.05% trypsin (Table 2). Incubate at 37 °C for 5 min.
12. Neutralize the trypsin by adding 4 mL of cold mESC media to the plate. Immediately use a 5 or 10 mL serological pipet to pipet up and down 15x in the plate to dissociate colonies into a single-cell suspension.
13. Transfer the suspension into a conical tube. Optionally, remove an aliquot to count cells using a hemocytometer.
14. Pellet the cells by centrifugation. Aspirate the supernatant.
15. Resuspend the cell pellet in fresh media. Plate 5 × 10⁶cells (typically 1:6 passaging ratio) in a fresh gelatinized 10 cm plate and incubate at 37 °C. Repeat steps 1.9–1.15 as needed until ready to proceed with transfection.
16. To make frozen cell stocks, trypsinize a plate of mESCs (as in steps 1.10–1.14). Resuspend the cell pellet in freezing media (250–500 µL per 5 × 10⁶ cells), aliquot into cryovials, and freeze slowly in a cell freezing container at -80 °C overnight before transferring to liquid nitrogen storage.

2. Transfection

NOTE: Perform transfection on a day when mESCs are ready for passaging. Plasmids may begin expressing as early as 3-5 h after transfection. Once expressing, we recommend handling cells in the dark and minimizing exposure to temperatures below 37 °C to prevent premature activation, since previous work has shown that Magnet optogenetic domains dimerize not only under blue light illumination but also upon exposure to cold³¹. Keep plates in a dedicated incubator or shield a shelf in a shared incubator from light with aluminum foil. When handling cells outside the incubator, turn off or block all room lights and windows beforehand, and use a red-light headlamp to see. Do not leave the incubator door open for longer than necessary. Take only small batches of plates out of the incubator at a time, and rest plates on an insulating surface (i.e., a clean styrofoam box lid) while handling to minimize contact with room-temperature work surfaces. Warm media fully before use. Do not inspect plates under a light-emitting microscope.

1. The day before transfection, gelatinize a 10 cm plate.
2. On the day of transfection, aspirate gelatin from the plate and add 7.5 mL of media to prewarm at 37 °C. Prewarm reduced-serum medium at 37 °C for resuspending cells.
3. Dilute 18.75 µL of the transfection reagent in 250 µL of reduced-serum medium. Tap gently to mix and incubate for 5 min at room temperature.
4. If co-expressing two plasmids encoding the N- and C-fragments, add 3.75 µg of each plasmid to 250 µL of reduced-serum medium. If only expressing one plasmid (i.e., a GFP negative control or a full-length BirA positive control), add 7.5 µg of the single plasmid instead.
5. Add the plasmid/reduced-serum medium mix to the transfection reagent/reduced-serum medium mix and tap/invert to mix. Incubate at room temperature for 15 min.
6. In the meantime, trypsinize and pellet mESCs (steps 1.10–1.14). Resuspend the pellet in warmed reduced-serum medium to a concentration of 10 × 10⁶ cells/mL.
7. Add 7.5 × 10⁶ (750 µL) resuspended cells to the plasmid/transfection reagent/reduced-serum medium mixture. Invert the tube to mix. Incubate for 10 min at RT, inverting the tubes occasionally to keep cells suspended.
8. Invert the tubes again briefly and add the mESC/plasmid/transfection reagent/reduced-serum medium mixture to the plate containing warmed media. Gently rock the plate to evenly distribute the cells.
9. Incubate the plate at 37 °C for 3–5 h, allowing the cells to adhere within this time.
   NOTE: From this point on, we recommend observing the above precautions against exposure to light and temperatures below 37 °C.
10. Gently aspirate the old media. Add fresh warmed media, pipetting down the side of the plate to avoid dislodging cells. Resume incubation at 37 °C.
11. If activating/lysing at 24 h after transfection, proceed to section 3. If planning to activate/lyse at 48 h after transfection, change media ~24 h after plating the transfected cells.

3. ALIBi activation and cell lysis

NOTE: Continue to handle cells/samples in the dark until the beginning of elution (step 5.4). Once cells have been chilled on ice (step 3.15), keep samples cold and use only chilled reagents until the beginning of elution. Ensure that working surfaces, equipment, reagents, and plastics are RNAse-free by using freshly opened items and wiping or rinsing hard surfaces with an RNAse decontaminating solution and 70% ethanol. Wear gloves whenever handling samples or equipment. For samples intended for MS proteomics, also minimize keratin contamination by keeping the work space dust-free; cover bare skin, and tie back long hair.

1. Prechill the centrifuge to 4 °C.
2. With lights off, move the transfected plates to a temporary 37 °C incubator so they will not be exposed to temperature fluctuation in the main incubator after the transilluminator is placed inside.
3. Wipe down the blue light transilluminator with 70% ethanol and connect it to power. Place the transilluminator in the main incubator and allow incubator conditions to re-equilibrate while continuing with the setup.
4. Warm an aliquot of media to be dosed with biotin and cycloheximide (CHX) (10 mL of media per 10 cm plate) to 37 °C. If running control samples without supplemental biotin, also warm a separate aliquot to be dosed with CHX only.
   NOTE: Some buffer components can be mixed the day before use and stored overnight at 4 °C (see Table 2). Note that different lysis and wash buffer recipes were used for experiments intended for western blot or MS proteomics and those intended for RNA sequencing.
5. Chill an aliquot of DPBS to be dosed with CHX (20 mL per 10 cm plate).
6. Prepare the RNAse removal buffer, salt wash, lysis buffer, and wash buffers (see Table 2). For buffers with dense/viscous components such as glycerol and Triton X-100, pipet the completed buffer to ensure even mixing. Chill buffers on ice prior to use.
   NOTE: For bead washing steps, briefly spin the tube containing beads in their current buffer in a pocket centrifuge to collect the contents at the bottom. Place the tube in a magnetic rack for 1 min or until beads have fully migrated. Aspirate the supernatant without touching the bead pellet. Without letting the bead pellet dry, add the next buffer. To resuspend the bead pellet, either pipet gently or roll the closed tube between one’s fingers while holding it against the magnetic rack, such that the beads are quickly pulled in multiple directions by the magnet. Finally, invert the resuspended beads several times to ensure the inside walls of the tube are washed.
7. Resuspend the stock bottle of streptavidin beads by gentle pipetting or rocking. Transfer 450 µL of beads to a protein low-binding tube for each 10 cm plate of mESCs.
8. Following the above NOTE on bead washing, discard the bead storage buffer and wash beads 2x with 900 µL of RNAse removal buffer.
   NOTE: Proceed to the next step without delay; extended time in RNAse removal buffer may decrease the beads’ binding capacity.
9. Wash beads 1x with 900 µL of salt wash.
10. Wash beads 2x with 900 µL of Wash I Buffer. After resuspending beads in the second round of Wash I buffer, store the beads in the Wash I buffer at 4 °C while performing activation and cell lysis.
11. Add biotin (50 µM final concentration; 1:1,000 dilution of 50 mM stock) and CHX (100 µg/mL final concentration; 1:1,000 dilution of 100 mg/mL stock) as appropriate to the warmed aliquot(s) of media. Keep at 37 °C until use.
12. Add CHX (100 µg/mL final concentration; 1:1,000 dilution of 100 mg/mL stock) to the cold aliquot of DPBS and keep chilled until use.
    NOTE: The following two steps describe activation of ALIBi via supplemental biotin and blue light illumination. An ideal duration of activation is long enough to yield an adequate amount of ribosome biotinylation for downstream assays, but short enough to preserve spatiotemporal resolution. Ideal activation time may vary based on parameters such as the cell type, scale of the experiment, expression level of the split enzymes, abundance of ribosomes near the organelle of interest, and sensitivity of the analysis technique. We recommend 15 minutes as a starting point, which is the activation time used in our representative results (Figure 2) and in our accompanying work³⁰.
13. Aspirate media from the transfected plates. Immediately add 10 mL of the warm media containing CHX ± biotin per plate.
14. For any negative control plates not to be illuminated, place them in a dark incubator. For plates to be illuminated, place them directly on top of the transilluminator, turn on the transilluminator, and incubate for 15 min.
15. When illumination is complete, immediately wash the plates 2x with 10 mL of cold DPBS + CHX per plate. Place the plates on ice between and after the washes. After aspirating the second wash, tilt the plate and aspirate any residual DPBS.
16. Keeping each plate as flat as possible on ice, proceed to lysis by adding 2 mL of ice-cold lysis buffer. Incubate plate on ice for 5 min, tapping plate to dislodge cells 2–3x during that time.
    NOTE: if kept dark under foil and on ice, plates can be moved out of the cell culture area to another dark space during this time.
17. Dislodge any remaining cells by using a micropipette to pipet the lysate up and down on the plate surface while avoiding formation of bubbles, then transfer lysate to a chilled and labeled protein low-binding tube.
    NOTE: OPTIONAL: For some difficult-to-lyse organelles such as the nucleus and nucleolus, sonication may help.
18. In the case of difficult-to-lyse organelles, sonicate all samples in the experiment for 45 s at 4 °C (power setting of 3 watts). If the sonicator design requires insertion of a probe into an open sample tube, transfer the lysate initially from the plate into a larger tube to prevent overflow during sonication; then, transfer it to a 2 mL tube.
19. Incubate the lysate on ice for 15 min.
20. Centrifuge the lysate at 17,000 × g for 3 min at 4 °C. Transfer the supernatant (clarified lysate) to a chilled protein low-binding tube without disturbing the pellet of cell debris.
    NOTE: If RNA-seq analysis is intended, an aliquot of the clarified lysate can now be collected as the total RNA fraction (see Supplemental File 1, “Recommendations for RNA sequencing” step 1.1) for details.
    NOTE: If the cell quantities, transfection/culture conditions, and lysis conditions have been uniform for all samples, we typically assume lysate concentrations are comparable between samples and proceed without quantifying total protein concentration. This minimizes the elapsed time between activation and affinity purification.

4. Affinity purification

1. Pellet the washed streptavidin beads using the magnetic rack. Aspirate the Wash I buffer.
2. Transfer 1,200 µL of clarified lysate to the tube containing the washed beads. Pipet gently to resuspend the beads. Store the remainder of the clarified lysate at -80 °C.
3. Incubate the lysate with beads at 4 °C in the dark for 1 h on the tube rotator.
4. During this incubation, prepare the TEV elution buffer (Table 2).

5. Elution

1. Following the notes on bead washing in section 3, pellet the beads on a magnetic rack and transfer the supernatant to a chilled protein low-binding tube. Store the supernatant at -80 °C.
2. Wash beads 2x with 900 µL of Wash I buffer.
3. Wash beads 1x with 900 µL of Wash II buffer.
   NOTE: After completion of this step, samples no longer need to be handled in the dark.
4. Add 450 µL of TEV elution buffer to the beads.
5. Incubate beads in TEV elution buffer for 1 h at room temperature on the tube rotator. For smaller-scale experiments using low volumes of TEV elution buffer, incubate the tubes on a shaker at 1,000–2,000 rpm instead.
6. Pellet the beads on the magnetic rack. Transfer the eluate to a protein low-binding tube. If proteomics analysis or western blot is intended, store the eluate at -80 °C. If RNA-seq analysis is intended, see Supplemental File 1 “Recommendations for RNA sequencing” step 1.2 for eluate storage instructions.
7. OPTIONAL: To preliminarily determine whether the affinity purification has yielded appreciable rRNA/mRNA (indicating that ribosomes have been isolated), analyze eluates on a microvolume spectrophotometer using TEV elution buffer as a blank.
   NOTE: Eluates from cells containing the activated system should have greater absorbance at 260 nm than eluates from cells lacking the split enzymes, blue light/biotin, or L31-FTA.

6. Western blot

NOTE: Until lysate and flow-through samples have been denatured in Laemmli buffer, we recommend continuing to avoid light exposure. We use precast gels, commercially available transfer buffer and running buffer, and a semi-dry transfer system (see the Table of Materials). SDS-PAGE and western blot are performed according to the manufacturers’ instructions for these components, unless otherwise stated below.

1. Add 6x Laemmli buffer to the lysate, flowthrough, and/or eluate samples to a final concentration of 1x Laemmli buffer. Heat at 95 °C in a heat block or thermocycler for 10 min. If condensation forms on the tube lid/walls, spin down tubes briefly.
2. Set up a gel with running buffer in the gel tank, per the tank manufacturer’s instructions. Load the samples and the ladder.
3. Run the gel at 125 V for 45–75 min until the dye front approaches the bottom of the gel. In the meantime, prepare PBST and blocking buffers (Table 2).
4. Perform a semi-dry transfer per the transfer system manufacturer’s instructions.
   NOTE: For all membrane washing/incubation steps, ensure that the membrane is fully submerged and place the membrane tray on an orbital or tilting rocker. For a 10 x 15 cm tray, this requires 25–50 mL of buffer.
5. Remove the membrane from the transfer stack and proceed to the next step immediately to ensure the membrane does not dry out.
   NOTE: OPTIONAL: Dry spots on the membrane can result in uneven blocking or probe binding, especially with probes that have short incubations such as streptavidin-horseradish peroxidase. To avoid this issue, we re-wet membranes after transfer in 100% methanol for 30 s, then in water for 5 min. This measure is similar to general recommendations for re-wetting PVDF membranes that have dried after transfer^32,33.
6. Wash the membrane in PBST for 3 x 10 min at room temperature.
7. Following the specified conditions in Table 3 for each western blot probe, perform the blocking, washing, probe incubation, and secondary antibody incubations.
8. After the final wash in step 6.7, mix the ECL substrate components per the manufacturer’s instructions and coat the membrane evenly with it. Capture the image on a chemiluminescence gel imaging system.

Results

Western blot (protocol section 6) is the most expedient way to preliminarily assess the success of an experiment. When membranes are probed with streptavidin-HRP, lysate from cells where all components of the system were present should contain a band corresponding to biotinylated L31-FTA (Figure 2A, lane 20). The same band should be present, usually at greater intensity, if cells were transfected with full-length BirA as a positive control (Figure 2A, lanes 1–4). This band should be fainter or absent in samples where one or more system components was omitted (Figure 2A, all other lanes). Since mammalian cells contain several endogenous biotinylated carboxylase proteins in the 70–80 and 120–130 kDa range^34,35, we typically see bands at these molecular weights in all lysate samples. Membranes can also be probed with anti-V5 antibody to confirm expression of the split BirA fragments.

Western blot of the lysate, flowthrough, and eluate samples can assess whether an affinity purification experiment was successful (Figure 2B). A decrease in biotinylated L31-FTA in the flowthrough relative to the lysate indicates that a substantial proportion of the biotinylated ribosomes were captured by affinity purification. Since streptavidin conjugates to AviTag distal to the TEV cleavage site on L31-FTA, the biotinylated portion of L31-FTA is cleaved during TEV-mediated elution and thus, no biotinylated L31-FTA should be present in the eluate. L31-FTA in the non-biotinylated, biotinylated, and TEV-cleaved states are all detectable by anti-FLAG antibody, which should reveal that the eluate contains a cleaved L31-FTA band at a slightly lower molecular weight than intact L31-FTA. Note that the L31-FTA bands in the lysate and flow-through will often have comparable intensity when probed with anti-FLAG antibody; since activation is performed briefly, only a small fraction of total L31-FTA is biotinylated and affinity-captured. Probing for other ribosomal proteins such as Rps12 demonstrates the presence of the small ribosomal subunit in the eluate, suggesting that affinity purification of biotinylated L31-FTA captures whole ribosomes. Conversely, probing with anti-V5 antibody shows that proteins that do not bind the ribosome, such as the split BirA fragments themselves, are absent from the eluate.

Determining the optimal bead:lysate ratio is important, and Figure 2C demonstrates the diminishing utility of increasing the amount of lysate beyond the capacity of the beads. In this experiment, 2.5-fold more lysate was used with the same 450 µL of streptavidin-coated beads. Consequently, a substantial amount of biotinylated L31-FTA remained in the flowthrough. This occurred in part because the endogenous biotinylated proteins also occupy binding sites on the streptavidin-coated beads, as demonstrated by their partial depletion in the flowthrough. The endogenous biotinylated proteins are not detectable in the eluate because they are not released from the beads during TEV-mediated elution, but they are recovered when post-elution beads are heated in Laemmli buffer.

Immunofluorescence images showing split enzyme localization to targeted organelles, as well as results from other system-based experiments such as RNA-seq and MS proteomics of eluates, can be found in our recent publication³⁰.

Figure 1: Schematic for ALIBi components and experiment workflow. (A) Role of each component during activation, affinity purification, and elution. (B) Position of FLAG-TEV-AviTag in the Rpl31 coding sequence, and position of Rpl31 on the structure of the ribosome (PDB 4V6X). (C) Structure of the N-fragment (pink) and C-fragment (purple) of split BirA enzyme, with the arrowhead denoting the split site (PDB 1BIB). (D) Examples of construct pairs used to target the split BirA enzyme fragments to organelles. (E) Timeline of a typical experiment. This figure was adapted from Zhang et al.³⁰. Abbreviations: ? = hypothetical copurifying proteins; ALIBi = AviTag-specific Location-restricted Illumination-enhanced Biotinylation; TEV = tobacco etch virus; MS = mass spectrometry; CDS = coding sequence; ER = endoplasmic reticulum; eMag = enhanced Magnet; NES = nuclear export sequence; MAVS = mitochondrial antiviral-signaling protein. Adapted with permission from Zhang et al.³⁰. Please click here to view a larger version of this figure.

Figure 2: Representative western blot results. (A) Biotinylation of L31-FTA, as detected by streptavidin-HRP, under conditions including different combinations of ALIBi components. anti-V5 antibody detects the N- and C-terminal fragments at lower and higher molecular weights, respectively. GAPDH serves as a loading control. Full-length BirA serves as a positive control. Lanes with GFP expressed in lieu of split enzymes, or with untagged mESCs, serve as negative controls. This panel was adapted from Zhang et al.³⁰ (B) Abundance of biotinylated L31-FTA (detected by streptavidin-HRP), total L31-FTA (detected by anti-FLAG antibody), split enzyme fragments (detected by anti-V5 antibody), and the ribosomal small subunit protein Rps12 in lysate, flowthrough, and eluate fractions. Percentages indicate the proportion of the total sample volume that was loaded. (C) Abundance of biotinylated L31-FTA, total L31-FTA, and split enzyme fragments in fractions from an affinity pulldown experiment in which 2.5x the amount of lysate was used with the standard 450 μL of streptavidin-coated beads. For lane 4, the beads after elution were heated at 95 °C for 10 min in 450 μL of 1x Laemmli buffer. Abbreviations: ALIBi = AviTag-specific Location-restricted Illumination-enhanced Biotinylation; HRP = horseradish peroxidase; GFP = green fluorescent protein; mESCs = mouse embryonic stem cells. Panel (A) is adapted with permission from Zhang et al.³⁰. Please click here to view a larger version of this figure.

Table 1: Subcellular localization sequences. Peptide sequences used to target the split enzymes to organelles of interest^{36,37,38,39,40,41}. Adapted with permission from Zhang et al.³⁰.Please click here to download this Table.

Table 2: Buffer recipes. Includes buffers used for cell culture, cell lysis, affinity purification/elution, and western blot. Please click here to download this Table.

Table 3: Western blot conditions. Includes probe/antibody dilutions and conditions for blocking, wash, and probe/antibody incubation steps. Please click here to download this Table.

Supplemental File 1: Example strategy for inserting FTA into an endogenous ribosomal protein gene via CRISPR. Please click here to download this file.

Supplemental Figure S1: Example CRISPR strategy for insertion of FLAG-TEV-AviTag into an endogenous ribosomal protein gene. Two PX459-based plasmids, which each express a Cas9 protein fused to T2A and a puromycin resistance protein, as well as a guide RNA, are transfected into wild-type mESCs to yield two Cas9-gRNAs. Simultaneously, a donor plasmid is also transfected. The Cas9-gRNAs then cut the genomic DNA at sites surrounding the stop codon and the donor plasmid to release a linearized dsDNA repair template. This allows for homology-directed repair of the genomic DNA. In the successfully edited genomic DNA, the original gRNA cut sites now harbor mutations to prevent re-cutting. Abbreviations: CRISPR = clustered regularly interspaced short palindromic repeats; TEV = tobacco etch virus; puroR = puromycin resistance protein; gRNA = guide RNA; mESCs = mouse embryonic stem cells; RP = ribosomal protein. Please click here to download this file.

Supplemental Figure S2: Recommended primer design for genotype screening of post-CRISPR clones. Configurations A and B are recommended primer placements, where at least one primer in each pair is distal to the 300 bp flanking regions. Configuration C is not recommended, due to the risk of a false positive from amplification of residual donor plasmid. Abbreviations: CRISPR = clustered regularly interspaced short palindromic repeats; gRNA = guide RNA; RP = ribosomal protein. Please click here to download this file.

Discussion

ALIBi offers several advantages relative to existing techniques for studying subcellular translation and has potential future applications in ribosome biology and beyond. This protocol requires similar or less time compared to sequential lysis and centrifugation^21,22 and is potentially applicable to any subcellular compartment that can be genetically targeted with a localizing peptide sequence. Compared to constitutively active BirA^10,24, this method does not require cells to be cultured in biotin-depleted media. Previously reported concentrations of biotin in fetal bovine serum range from 0.04 µM to 0.2 µM^25,42,43; thus, our mESC media, which contains 15% fetal bovine serum and no other biotin-containing components, has an estimated biotin concentration of 0.006–0.03 µM. Compared to promiscuous proximity biotinylation methods^26,27,28, this approach isolates subcellular translational machinery rather than subcellular proteomes. Additionally, it allows for non-denaturing elution, such that the eluted translational machinery is theoretically suitable for downstream assays requiring native complexes.

The utility of this method has been illustrated by our recent work³⁰, in which the seminal use of the approach enabled an unprecedented comparison of ribosome subpopulations in mammalian cells via characterization of associated mRNAs and ribosome-associated proteins. Future applications may extend to other subcellular compartments where localized translation has been described. Other translational machinery features, such as post-translational modifications, RP splicing isoforms, and rRNA modifications also remain to be explored at the subcellular level. Beyond the additional applications towards ribosome biology, ALIBi, in principle, could also be used to study subcellular populations of other protein complexes.

This method, however, harbors some limitations and challenges. First, it requires the insertion of FTA into a ribosomal protein and the expression of the split enzyme constructs, which could prove difficult in model systems with limited means for genetic manipulation. It also requires working under dark conditions for part of the protocol and maintaining temperature control of samples to prevent premature activation. It is thus critical to minimize exposure to light and temperatures below 37 °C after transfection and prior to activation, and to proceed through the activation, lysis, and affinity purification without delay. Once chilled during lysis, samples should not be allowed to warm above 4 °C to minimize degradation and continued BirA activity. 

Troubleshooting may be required if the expected degree of L31-FTA biotinylation is not observed. If no biotinylation is detected in lysates after activation, first confirm that all components are present (L31-FTA, split BirA N- and C-fragments, supplemental biotin, blue light), and check that biotinylation is detected in a positive control sample expressing full-length BirA. Additionally, in our hands, mESCs express sufficiently high levels of endogenous biotinylated proteins (Figure 2B, C) that these can serve to confirm that streptavidin-HRP is appropriately detecting biotinylation. Consider increasing the duration of activation, the amount of lysate loaded onto SDS-PAGE, and the exposure time for Western blot chemiluminescence detection. If using a previously untested FTA-tagged cell line or organelle-targeted split enzyme, consider whether the FTA-bearing portion of the ribosome has sufficient interaction with the split enzyme for biotinylation to occur.

Conversely, observing substantial biotinylation in non-activated control samples would also be an undesirable outcome because it would suggest that AviTag biotinylation is occurring outside the activation period. Since ribosomes and pre-ribosomes traffic between subcellular compartments (i.e. between the nucleolus and nucleus to the cytoplasm during biogenesis^3,4, or from the cytoplasm to the ER via signal recognition particle-mediated docking to the translocon ⁵), ribosomes that were biotinylated prior to the activation/lysis timepoint may no longer be localized to the organelle of interest and thus may no longer have organelle-specific characteristics. If background biotinylation occurs despite minimal exposure to light and temperature excursions, consider decreasing the amount of split enzyme plasmid transfected and investigate whether any media components contain a higher-than-anticipated level of biotin. While elimination of all background biotinylation prior to activation may be infeasible, in our experience, a five-fold increase in biotinylation upon activation is sufficient for isolating distinct ribosome subpopulations³⁰. 

The affinity purification process may also require troubleshooting, which typically involves optimizing the bead:lysate ratio or the stringency of the washes. Note that in lysates with a large quantity of biotinylated ribosomes (i.e., full-length BirA or cytosol-targeted split BirA), uncaptured biotinylated L31-FTA may remain in the flowthrough. Pulldown of all biotinylated ribosomes is not always necessary to obtain sufficient material for downstream analysis.

Finally, one may encounter a lack of anticipated differences upon comparison of two or more subcellular ribosome populations by RNA sequencing or MS proteomics. We recommend using immunofluorescence to check whether both split enzyme fragments are sufficiently localized to the desired subcellular compartment. Consider testing other organelle-targeting sequences. Isolation of organelle-localized ribosomes may also be hampered by insufficient organelle lysis; consider testing other lysis detergents or sonication. Organelle-specific differences in translational machinery may also be obscured if there is an abnormally high level of split BirA activity prior to activation or after lysis, resulting in non-specific ribosome biotinylation; see above for measures to reduce undesired biotinylation. Similarly, performing the activation for too long could allow organelle-localized ribosomes to migrate away and lose their organelle-specific characteristics prior to cell lysis. Additionally, consider decreasing the stringency of washes to avoid stripping organelle-specific ribosome-associated molecules.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
-80 °C storage			For cell culture
0.1% gelatin in water	MilliporeSigma	ES-006-B	For cell culture
0.2 µm filter units	Fisher	04-977-158	For cell culture
0.5% trypsin-EDTA (10X)	ThermoFisher	15400-054	For cell culture
1 M MgCl2	ThermoFisher	AM9010	For lysis & affinity purification
1 M Tris pH 7.5	ThermoFisher	15567027	For lysis & affinity purification
1 M Tris pH 8	ThermoFisher	AM9855G	Optional: for inserting FTA into an endogenous ribosomal protein locus
1.5 and 2 mL microcentrifuge tubes, autoclaved			For transfection
1.5, 2 mL, and 5 mL protein low-binding microcentrifuge tubes	Eppendorf	022431081, 022431102, 0030108302	For lysis & affinity purification
10 cm tissue culture plates			For cell culture
10 M NaOH	MilliporeSigma	72068-100ML	For lysis & affinity purification
10x Tris/glycine/SDS running buffer concentrate	Bio-Rad	1610772	For Western blot
37 °C tissue culture incubator x 2			For cell culture. One of these serves as the main incubator for cell culture and illumination of transfected cells. Temporary space is needed in a second incubator on the day of ALIBi activation to house transfected cells while setting up the transilluminator in the main incubator.
37 °C water bath			For cell culture
5 M NaCl	ThermoFisher	AM9010	For lysis & affinity purification
50 mM biotin	ThermoFisher	B20656	For lysis & affinity purification
6x Laemmli buffer	FIsher	50-196-784	For Western blot
70% ethanol			For disinfection
Acetonitrile	Fisher	A955	Optional: for mass spectrometry sample preparation
AcTEV protease kit	ThermoFisher	12575015	For lysis & affinity purification
ALIBi C-fragment plasmid	Addgene	#235615, 235636 – 235641	Midi-prep scale is required for transfection
ALIBi N-fragment plasmid	Addgene	#235614, 235635, 235642, 235643	Midi-prep scale is required for transfection
Aluminum foil			For lysis & affinity purification
ammonium bicarbonate	MilliporeSigma	5.3305	Optional: for mass spectrometry sample preparation
anti-FLAG antibody	MilliporeSigma	F3165	For Western blot
anti-GAPDH antibody	Cell Signaling	5174	For Western blot
anti-mouse secondary antibody	GE Healthcare	NA931V	For Western blot
anti-rabbit secondary antibody	GE Healthcare	NA934V	For Western blot
anti-Rpl12 antibody	Abcam	ab175219	For Western blot
anti-Rpl31 antibody	Abcam	ab103991	For Western blot
anti-Rpl5 antibody	ProteinTech	15430-1-AP	For Western blot
anti-V5 antibody	ThermoFisher	R960-25	For Western blot
Apyrase	NEB	M0398L	For lysis & affinity purification
Beta-mercaptoethanol (55 mM)	ThermoFisher	21985-023	For cell culture
Blue light transilluminator	Clare Research Chemical	DR46B (small) or DR196 (large)	For lysis & affinity purification
Bovine serum albumin (BSA)	Fisher	BP1600-100	For Western blot
C18 tips (OMIX C18 tips)	Agilent	A57003100	Optional: for mass spectrometry sample preparation
Cell freezing container			For cell culture
Centrifuge fitted for 15 mL and/or 50 mL conical tubes			For cell culture
Centrifuge for 1.5-2 mL microcentrifuge tubes			For lysis & affinity purification. Should be capable of refrigeration down to 4 C and speeds up to 17,000 g. Ideally, it is located in a room that can be temporarily darkened while transferring tubes in and out.
Clarity ECL kit	Bio-Rad	170-5061	For Western blot
Criterion gel tank	Bio-Rad	1656001	For Western blot
Criterion TGX pre-cast gels, 4-20%	Bio-Rad	5671095	For Western blot. Additional well sizes and layouts available
Cycloheximide	MilliporeSigma	C7698-5G	For lysis & affinity purification
Deoxycholate	MilliporeSigma	S1827	For lysis & affinity purification
Dithiothreitol	ThermoFisher	PIA39255	Optional: for mass spectrometry sample preparation
DPBS, no calcium, no magnesium	ThermoFisher	14190-250	For cell culture
ES-qualified FBS	MilliporeSigma	ES-009-B	For cell culture
Ethanol	Gold Shield	412828	For Western blot
Ethanol	MilliporeSigma	459828	Optional: for mass spectrometry sample preparation
Formic acid	MilliporeSigma	A117	Optional: for mass spectrometry sample preparation
Fragment size analyzer and consumables		Agilent	Optional: for RNA sequencing library preparation. Agilent 2100 Bioanalyzer
Freezing media	ThermoFisher	12648-010	For cell culture
Gel imaging system with chemiluminescence			For Western blot
Gel loading tips			For Western blot
Gel tank power source			For Western blot
Glycerol	MilliporeSigma	G5516-100ML	For lysis & affinity purification
guanidinium-phenol reagent (TRIzol)	ThermoFisher	15596-018	Optional: for RNA sequencing library preparation
guanidinium-phenol reagent for large sample volume (TRIzol LS)	ThermoFisher	10296010	Optional: for RNA sequencing library preparation
guanidinium-phenol RNA extraction kit (DirectZol kit)	Zymo	R2060	Optional: for RNA sequencing library preparation
HALT Protease Inhibitor	ThermoFisher	78425	For lysis & affinity purification
Hemocytometer			For cell culture
Heptafluorobutyric acid	ThermoFisher	52411	Optional: for mass spectrometry sample preparation
High pH fractionation kit	ThermoFisher	84868	Optional: for mass spectrometry sample preparation
Hydroxylamine	MilliporeSigma	467804	Optional: for mass spectrometry sample preparation
IGEPAL CA-630	MilliporeSigma	I8896	Optional: for inserting FTA into an endogenous ribosomal protein locus
Implementation of guide RNA scoring algorithm	Benchling	https://www.benchling.com/	Optional: for selecting guide RNAs to insert the FTA sequence into an endogenous ribosomal protein locus. User-friendly interface that implements the guide RNA scoring algorithm from Doench, J.G. et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nature Biotechnology. 34 (2), 184–191, doi: 10.1038/nbt.3437 (2016).
Instant milk powder, nonfat	Safeway	116100001	For Western blot
Insulating surface, i.e. styrofoam box lid			For transfection
Iodoacetamide	ThermoFisher	A39271	Optional: for mass spectrometry sample preparation
Knockout DMEM	ThermoFisher	10829-018	For cell culture
L-Glutamine (100x)	MilliporeSigma	TMS-002-C	For cell culture
L31-FTA mouse embryonic stem cells			Available on request from the Barna Lab, mbarna@stanford.edu
Light microscope with 4-10x magnification			For cell culture
Lipofectamine 2000	ThermoFisher	11668-019	For transfection
Liquid nitrogen storage			For cell culture
LysC	Wako	125-05061	Optional: for mass spectrometry sample preparation
Magnetic rack for 1.5-2 mL tubes	ThermoFisher	CS15000	For lysis & affinity purification
Methanol	VWR	EM-MX0475-1	For Western blot
Microcentrifuge tube rotator			For lysis & affinity purification
Micropipettes and sterile or autoclaved micropipette tips			For cell culture
Microseal for 96-well plates	Bio-Rad	MSB1001	Optional: for inserting FTA into an endogenous ribosomal protein locus
Microvolume spectrophotometer	ThermoFisher	NanoDrop 2000	Optional
midi-prep kit (Plasmid Plus Midi Kit)	Qiagen	12945	Optional: for inserting FTA into an endogenous ribosomal protein locus
mLIF (10,000 U/µL)	Gemini	400-495 10^7	For cell culture
Multiplex indices kit (NEBNext Multiplex Oligos for Illumina (Index Primers Sets 1 and 2))	NEB	E7335S, E7500S	Optional: for RNA sequencing library preparation
MyOne C1 streptavidin Dynabeads	ThermoFisher	65002	For lysis & affinity purification
No-weigh DTT	ThermoFisher	PIA39255	For lysis & affinity purification
Non-essential amino acids (100x)	MilliporeSigma	TMS-001-C	For cell culture
Non-expressing plasmid backbone (TOPO vector kit)	ThermoFisher	450031	Optional: for inserting FTA into an endogenous ribosomal protein locus
Orbital or tilting rocker			For Western blot
PBS powder	Fisher	BP661-10L	For Western blot
pCAGEN	Addgene	11160	Optional: for designing additional organelle-targeted ALIBi split enzyme constructsd
PCR thermocycler tubes			For Western blot
Penicillin/Streptomycin (100x)	ThermoFisher	15140163	For cell culture
pH strip	Fisher	8881-1	Optional: for mass spectrometry sample preparation
Plastic trays for membrane incubations			For Western blot
Pocket centrifuge for 1.5-2 mL centrifuge tubes			For lysis & affinity purification
Pocket centrifuge for PCR tubes			For Western blot
poly(A) mRNA isolation kit (NEBNext Poly(A) mRNA Magnetic Isolation Module)	NEB	E7490	Optional: for RNA sequencing library preparation
Polymerase master mix suitable for genotyping (MyTaq Hot Start Red Mix)	BioLine	BIO-25048	Optional: for inserting FTA into an endogenous ribosomal protein locus
Protein ladder	ThermoFisher	26617	For Western blot
protein precipitation kit (ProteoExtract kit)	MilliporeSigma	539180	Optional: for mass spectrometry sample preparation
Proteinase K	MilliporeSigma	3115879001	Optional: for inserting FTA into an endogenous ribosomal protein locus
Purified water			For Western blot. i.e. from Milli-Q (MilliporeSigma) filtration system
puromycin	MilliporeSigma	P8833	Optional: for inserting FTA into an endogenous ribosomal protein locus
PX459	Addgene	62988	Optional: for inserting FTA into an endogenous ribosomal protein locus
Red-light headlamp			For transfection
Reduced-serum medium (OptiMEM)	ThermoFisher	11058021	For transfection
RNA column kit (RNA Clean & Concentrator-5 kit)	Zymo	R1016	Optional: for RNA sequencing library preparation
RNA library prep kit (NEBNext Ultra II Directional RNA Library Prep kit)	NEB	E7760S	Optional: for RNA sequencing library preparation
RNA non-stick 1.5 mL microcentrifuge tube	ThermoFisher	AM12450	Optional: for RNA sequencing library preparation
RNAseZap	ThermoFisher	AM9784	For lysis & affinity purification
Semi-dry transfer kit containing 5x transfer buffer, PVDF membrane, and blotting stacks	Bio-Rad	1704272	For Western blot
Semi-dry transfer system	Bio-Rad	1704150	For Western blot
Shaker for 1.5-2mL microcentrifuge tubes			For lysis & affinity purification. Should be capable of speeds up to 1000-2000 rpm
Sonicator (Microson Ultrasonic Cell Disruptor XL)	Misonix	XL-2000	For lysis & affinity purification
Sterile 15 mL and 50 mL conical tubes			For cell culture
Sterile cryovials, 1.5-2 mL capacity			For cell culture
Sterile serological pipettor and pipettes			For cell culture
Streptavidin-HRP	ThermoFisher	N100	For Western blot
SUPERase RNAse Inhibitor	ThermoFisher	AM2696	For lysis & affinity purification
SuperSignal West Femto Maximum Sensitivity Substrate kit	ThermoFisher	34095	For Western blot
Thermocycler or heat block			For Western blot
Thiourea	MilliporeSigma	T8656	Optional: for mass spectrometry sample preparation
Tissue culture hood			For cell culture
Tissue culture space and bench space in room(s) that can be darkened			For lysis & affinity purification
TMT 6plex labels	ThermoFisher	90066	Optional: for mass spectrometry sample preparation
triethylammonium bicarbonate	ThermoFisher	T7408	Optional: for mass spectrometry sample preparation
Triton X-100	MilliporeSigma	T8787-250ML	For lysis & affinity purification
Trypan Blue (0.4%)	Life Technologies	15250-061	For cell culture
Trypsin	ThermoFisher	90057	Optional: for mass spectrometry sample preparation
Turbo DNAse	ThermoFisher	AM2238	For lysis & affinity purification. Optional: for RNA sequencing library preparation
Tween-20	MilliporeSigma	P9416	For Western blot
Ultrapure water	ThermoFisher	10977023	For lysis & affinity purification
Urea	MilliporeSigma	U6504	Optional: for mass spectrometry sample preparation