High-throughput identification of nuclear envelope protein interactions in Schizosaccharomyces pombe using an arrayed membrane yeast-two hybrid library
Posted on: 7 September 2020
Preprint posted on 30 July 2020
Article now published in G3: Genes|Genomes|Genetics at http://dx.doi.org/10.1534/g3.120.401880
Categories: biochemistry, cell biology
Felix Mikus and Gautam Dey
Context
The analysis of integral membrane proteins has largely been limited to low-throughput methods, namely electron and super-resolution microscopy. The characterisation of membrane protein interactomes has been especially difficult due to their hydrophobic nature. The introduction of membrane yeast two-hybrid (MYTH) technology provided the first opportunities to characterise membrane protein localisation and interactions in high-throughput fashion [1]. By tagging bait proteins with the C-terminus of ubiquitin (Cub) and a transcription factor, interaction with the N-terminus of ubiquitin (Nup) tagged preys reconstitutes the ‘pseudo-ubiquitin’, following which the transcription factor is released by ubiquitin-specific-processing proteases (Fig. 1 A). Other high throughput approaches included the use of split-GFP in S. cerevisiae to assess the localisation of membrane proteins that assisted in the identification of several hundred putative INM proteins, a list that has further been expanded by the discovery of the INMAD pathway [2-4]. Based on these two previous advances, Varberg et al. [5] set out to assist in the characterisation of nuclear envelope protein interactomes by designing an arrayed library covering most integral membrane proteins.
Key outcomes
The authors utilised MYTH in S. pombe to screen a prey library consisting of 264 soluble and peripheral and 773 putative integral membrane proteins and, using automated measurements of colony density, enabled a high-throughout identification of both strong and weak interactions. Mapping the interactome of three model baits, the INM proteins Ima1, Cut11, and Lem2, demonstrated the potential of this library by identifying several known and novel interactors. Interestingly, prey interacting with Ima1 (Samp1/NET5) points towards a, as of now, uncharacterised role in lipid biogenesis and encourages in detail studies of the fungal and mammalian orthologs. The authors further used the library to compare the interactome of several alleles of Cut11. They convincingly showed that mutations of the C-terminus reduce interactions with NPC and SPB residing proteins, while not abolishing its localisation to said structures and propose a mechanism, in which Tts1 anchors Cut11 to the INM an NPC, while Sad1 might support its role at the SPB. The generated library will further present a great tool for future studies as it does offer a great coverage of most integral membrane proteins.
Questions for the authors
Given the localisation of some proteins to e.g. Ima1 is found at the SPB only during mitotic entry, would this screen be specific enough to pick up interactions during these short time points?
Can the prey library be tested with a positive control (Ost1-Cub) to assess the number of functional proteins in a way similar to the bait confirmation? This might explain the lack of known interactors.
Can you speculate about reasons why some proteins are only functional as bait but not as prey and vice versa?
How might secondary structures or modes of membrane association of the tested proteins affect the assay? Did you notice any changes in localisation in some of the tagged constructs?
You can read the authors’ response to these queries below – we would like to thank them for their detailed replies!
References
- Stagljar I, Korostensky C, Johnsson N, Te Heesen S. A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc Natl Acad Sci U S A. 1998;95(9):5187-5192. doi:10.1073/pnas.95.9.5187
- Smoyer CJ, Katta SS, Gardner JM, et al. Analysis of membrane proteins localizing to the inner nuclear envelope in living cells. J Cell Biol. 2016;215(4):575-590. doi:10.1083/jcb.201607043
- Khmelinskii A, Blaszczak E, Pantazopoulou M, et al. Protein quality control at the inner nuclear membrane. Nature. 2014;516(7531):410-413. doi:10.1038/nature14096
- Foresti O, Rodriguez-Vaello V, Funaya C, Carvalho P. Quality control of inner nuclear membrane proteins by the Asi complex. Science (80- ). 2014;346(6210):751-755. doi:10.1126/science.1255638
- Varberg JM, Gardner JM, Mccroskey S, Saravanan S, Bradford WD, Sue L. High-throughput identification of nuclear envelope protein interactions in Schizosaccharomyces pombe using an arrayed membrane yeast-two hybrid library. 2020.
doi: https://doi.org/10.1242/prelights.24487
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