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The assembly of proteins into defined complexes drives a plethora of cellular activities. These protein complexes often have a set of more stably interacting proteins as well as more unstable or transient interactions. Studying the in vivo components of these protein complexes is challenging as many of the techniques used for isolation result in the purification of only the most stable components and the transient interactions are lost. A technology called transient isotopic differentiation of interactions as random or targeted (transient I-DIRT) has been developed to identify these transiently interacting proteins as well as the stable interactions. Described here are the detailed methodological approaches used for a transient I-DIRT analysis of a multi-subunit complex, NuA3, that acetylates histone H3 and functions to activate gene transcription. Transcription is known to involve a concert of protein assemblies performing different activities on the chromatin/gene template, thus understanding the less stable or transient protein interactions with NuA3 will shed light onto the protein complexes that function synergistically, or antagonistically, to regulate gene transcription and chromatin remodeling.
To identify networks of stable and transient protein–protein interactions, technology termed transient isotopic differentiation of interactions as random or targeted (transient I-DIRT) was developed (Fig. 1) (1) . This technique combines mild in vivo chemical cross-linking and isotopic-labeling with mass spectrometric readout to classify proteins co-purifying with an affinity-tagged “bait” protein as stable, transient, or nonspecific. There has been a multitude of protein–protein interaction studies using in vitro chemical cross-linking; however, there have only been a few using in vivo cross-linking with affinity purification followed by mass spectrometric analysis (1-3). Here, we describe the methodology for transient I-DIRT as it provides for a straightforward and quantifiable approach for defining the in vivo stable and less stable protein interactions. As an example of transient I-DIRT, we describe an analysis of the multi-subunit complex NuA3. NuA3 is a histone acetyltransferase in Saccharomyces cerevisiae that acetylates lysine 14 of histone H3 and activates gene transcription (4) . NuA3 is a five member protein complex composed of Sas3, Nto1, Yng1, Eaf6, and Taf30 (4) . Only Yng1 and Nto1 are found solely in the NuA3 complex. To identify the stable and transient protein–protein interactions with NuA3 specifically, the Yng1 protein was utilized as the purification “bait.” Specifically a TAP-tagged version of Yng1 was used in the methods described; however, other affinity tags can be utilized with an appropriate antibody for enriching.
The methodological workflow for a transient I-DIRT analysis is shown in Fig. 1 . The key components of this methodology are (1) a strain with an affinity-tagged protein and (2) a strain without the tag on the protein that is grown isotopically heavy. The tagged and nontagged strains are grown to equivalent densities, subjected to a mild in vivo chemical cross-linking with formaldehyde, and frozen independently. The mild cross-linking helps to partially stabilize the protein interactions. Particularly for chromatin-associated protein complexes, the cross-linking has to be mild as extensive cross-linking precludes the efficient purification of protein complexes (1, 5) . Frozen strains are mixed 1:1 prior to cryolysis with a ball mill maintained under liquid nitrogen temperature, which provides a method for generating cell lysate without thawing. The ability to differentiate stable, transient, and nonspecific protein interactions with the affinity-tagged complex occurs at the point of thawing the cell lysate and extends for the duration of the purification procedure. During the course of the purification, the stable protein interactions (which are exclusively isotopically light) with the affinity-tagged complex are maintained, while the transient protein interactions (which are also isotopically light) are only somewhat maintained relative to their affinity for the tagged protein complex. Thus, for a transient protein interaction, some exchange of the isotopically light protein will occur with the isotopically heavy counterpart. For nonspecific protein associations with the affinity-tagged protein complex that occur during the purification procedure, there is an equal probability that the nonspecific protein will be isotopically light or isotopically heavy.
The ultimate readout for these stable, transient, and nonspecific protein interactions with the affinity-tagged protein complex is mass spectrometry. When peptides are assigned to a given protein co-purifying with the affinity-tagged complex, the type of protein interaction can be classified as one of the following: stable if the peptides are ~100% isotopically light, nonspecific if the peptides are ~50% isotopically light and ~50% isotopically heavy or transient if the peptides are in between 50 and 100% isotopically light. The following transient I-DIRT methodology is presented for an analysis of the NuA3 histone acetyltransferase, but the methodology can be extended to any affinity-tagged protein. Using the transient I-DIRT technique, we previously identified five stable (Sas3, Nto1, Yng1, Taf30, Eaf6), five transient (Pob3, Nap1, Spt16, Rsc8, Rsc7), and 278 nonspecific protein interactions with an NuA3 purification (1) . The analysis of NuA3 demonstrates the high level of nonspecific associations with affinity purifications, and the need for a technique like transient I-DIRT to identify the subpopulation of specific and transient interactions for subsequent functional studies.
Cryogenic lysis with a mixer mill is the preferred method for lysing and blending the cells. One should avoid methods, such as lysis, with glass beads as the samples will thaw during the procedure, which precludes uniform blending of the samples prior to thawing. If a mixer mill is not readily available, a reasonable alternative for cryogenic lysis is manual grinding of the cells in the presence of liquid nitrogen with a mortar and pestle. When manually grinding, the cells should be covered with liquid nitrogen during the lysis process. The cells should be ground into a fine powder. Grinding should continue until >75% lysis is visually observed with a light microscope. After the cells are ground, the cells are stored at −80°C as described above immediately after allowing the liquid nitrogen to evaporate.
Funding was provided by NIH grants P20RR015569, P20RR016460, and R01DA025755.
1The methodology presented is for incorporation of isotopically heavy arginine using the S. cerevisiae BY4741 arg4::KAN strain. If one is using a different auxotroph, heavy amino acid(s) or organism, then the incorporation efficiency of the heavy amino acid must be measured. For the transient I-DIRT procedure, one needs to approach complete incorporation of the heavy amino acid. To measure incorporation of heavy amino acid, the strain under study should be grown to mid-log phase and the proteins isolated. Proteins should be subjected to high resolution mass spectrometric analysis of tryptic peptides using a proteomics facility. Tryptic peptides containing the heavy amino acid should be completely labeled. It is always a good idea to label arginine or lysine as these are represented in tryptic peptides (since trypsin cuts after arginines and lysines). Additionally, it is a good idea to use isotopically heavy versions of carbon that are at least six Daltons heavy because (1) carbon isotopes (relative to deuterated) of amino acids do not affect peptide elution from reverse phase columns that are a key component of most mass spectrometric setups and (2) peptides with an amino acid that is less than six Daltons heavy result in isotopic overlap when compared to their isotopically light counterparts.
2The procedure outlined for affinity purification was optimized for NuA3. NuA3 is a relatively low abundance chromatin-associated protein complex. The amount of cell lysate necessary for other protein complexes must be determined empirically. The amount of cell lysate used in this section is a good starting point for most protein complexes that have been studied with the transient I-DIRT technique. Note that all steps in this section are scalable to the amount of starting lysate (e.g., 4 mg of coupled Dynabeads are used for each gram of cell powder).
3The affinity purification buffer described in this section is an empirically determined buffer that provides good yields for chromatin-associated protein complexes. The components of the buffer can be varied in accordance to the protein complex under study. Some of the components typically varied include: NaCl (200–400 mM) and TritonX-100 (0–1% v/v).
4The time of incubation with coated Dynabeads is empirically determined. Shorter affinity isolation times (e.g., 1–4 h) can be explored and may result in fewer nonspecific interactions (6).
5The mass spectrometric processing and data analysis are standard procedures for a proteomics facility. Proteomics facilities are available at many universities and are also available commercially.