Methods that could be used to identify protein interactors

There are several methods that could be used to identify protein interactors. There are advantages and disadvantages to each technique. For this project the most important factors were: a lack of bias when finding interactors and a tech- nique that required no prior knowledge of interactors. A brief explanation of varying methods that can be used to identify protein interactors are detailed be- low. Tandem affinity purification and chemical crosslinking were not used in this project, but are included to provide background on alternative techniques.

Co-immunoprecipitation

This method involves using an antibody against either the protein of interest, or a tag on the protein of interest itself. The antibody is attached to agarose beads, allowing the protein of interest, and any interactors, to be centrifuged down. The proteins that are pulled down can then be identified using mass spectroscopy. The benefits of using a tag, is that a commercial anti-tag antibody can be used, as opposed to a protein-specific and more expensive one. The disadvantage of a tag however, is that it can block interactions through steric hindrance and potentially interfere with protein expression. Examples of tags that can be used include: Fluorescent proteins (GFP, RFP), HA tag, FLAG tag or myc tag. This method does not need any prior knowledge of interactors, and can be used to find

Figure 2.2: Network of reticulon interactions. This highlights the interactions between RTN1, RTN3, RTN4, RTN5 and RTN13. A) RTN1 can be seen interacting with RTN2 and RTN4. RTN13 interacts with RTN4 and RTN3. B) RTN4 has 70 interactions with a plethora of different proteins. Among these are several reticulons, but also a lot of RAB proteins. The lines connecting the circles indicate a published interaction. The colour of the circles denotes their proposed cellular location: red = ER, pink = cytoplasm, yellow = vacuole, green = chloroplast, purple = peroxisome, blue = nucleus, orange = plasma membrane, brown = Golgi and grey = unknown location. (Full Colour Key: Appendix figure 7.2). This is taken from the Arabidopsis Interactions Viewer (Geisler-Lee et al., 2007).

transient interactions, due to not needing to purify the proteins.

This was the method chosen to identify interactors with RTN13, since a stable expression line of 35S:YFP-RTN13 had previously been created. Although the protein is not expressed under a native promoter, it allowed a quick method for identifying protein interactors.

Tandem affinity purification (TAP)

TAP is similar to co-immunoprecipitation, but involves two steps of purification, it was first used for yeast proteins (Rigaut et al., 1999). The tag on the protein of interest consists of: A calmodulin binding peptide, a TEV (Tobacco etch virus) protease cleavage site and Protein A. In the first purification step IgG antibodies (attached to agarose beads) bind to Protein A. The sample is washed, to remove non-specifically bound proteins, and then a TEV protease is added. This cleaves the tag in two. A second bead, with calmodulin protein, is added and binds to the second half of the tag. The protein of interest with its interactors, can be analysed through mass spectrometry. The number of washes makes this method unsuitable for transient interactions. As with co-immunoprecipitation, the large tag can interfere with protein interactions. The tag could also be inaccessible to the binding proteins, meaning the protein of interest is not ‘caught’ in the first place. One group has has recently used TAP and mass spectroscopy with both

A. thaliana seedlings and cell cultures to identify protein complexes (Van Leene et al., 2015).

Chemical crosslinking

Chemical crosslinking can be performedin vivo orin vitro, and it involves chem- ically binding two proteins together (reviewed in (Sinz, 2006; Bruce, 2012)). This technique has been used for a long time to find protein-protein interactions even in plants (Baird and Hammes, 1976). This technique is still being used and de- veloped and indeed groups are cross-linking proteins in planta to find interactors (Zhu et al., 2016). Once the proteins are cross-linked, these proteins can be di- gested and the fragments analysed by mass spectrometry. There will then be fragments containing material from different proteins, which enables the user to find not only which proteins are interacting, but where they interact as well. This method can find interactors expressed at native levels. There is no risk of steric hindrance as there are no tags. It would however, be difficult to have an appro- priate control as proteins nearby, but not interacting, can be cross-linked.

2.2 Aims and approach

The first aim of this project was to identify any proteins that could be found to be interacting with RTN13. A stable expression of YFP-RTN13 under an enhanced 35S promoter in A.thaliana was used and developing seed was collected. As shown in the gene expression heat maps in figure 1.9, this is when RTN13 is primarily expressed. By collecting ‘green’, or developing seed, the proteins that are interacting with RTN13 are specific to this developmental stage. The tissue was homogenised and co-immunoprecipitation was performed using an antibody against the YFP tag. This enabled RTN13, and any interacting proteins to be pulled down. The samples were sent for mass spectrometry analysis, in order to identify the proteins that were present in each sample. Controls were used to exclude any non-specific proteins.

Aims

• To find proteins that interact with RTN13, through mass spectrometry

• To clone these proteins and transiently express inN. benthamiana to iden- tify protein localisation