Menu

Close

High-throughput identification of nuclear envelope protein interactions in Schizosaccharomyces pombe using an arrayed membrane yeast-two hybrid library

Joseph M. Varberg, Jennifer M. Gardner, Scott McCroskey, Snehabala Saravanan, William D. Bradford, Sue L. Jaspersen

Preprint posted on July 30, 2020 https://www.biorxiv.org/content/10.1101/2020.07.29.227819v1

Behind the MYTH: the S.pombe nuclear envelope interactome

Selected by Gautam Dey

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. 

 

Figure 1: Taken directly from Fig. 1 A&B and Fig.2 A. of Varberg et al. 2020 under a CC-BY 4.0 international license.
(A) Schematic of the split-ubiquitin MYTH approach. Full-length integral membrane bait proteins are fused to the C-terminus of ubiquitin (Cub) and a LexA-VP16 transcription factor reporter (TF). Upon interaction with a prey protein fused to N-terminus of ubiquitin (Nub), the ubiquitin molecule is reconstituted and cleaved by proteases to release TF for expression of HIS3 and ADE2 reporter genes. (B) Diagram of S. pombe MYTH prey library composition by protein feature, conservation status and GO Compartment.
(C) Overview of MYTH library screening. Strains expressing the MYTH bait and prey are grown on PlusPlates, mated and diploids are selected by growth on SD-LT media in 96-spot format. Positive bait-prey interactions are identified by monitoring colony growth on selective media (SD-LTAH) supplemented with 3-AT. Colony densities are extracted and prey identity is assigned based on known prey library plate layout in an automated fashion. Figure 1: Taken directly from Fig. 1 A&B and Fig.2 A.

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

  1. 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
  2. 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
  3. 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
  4. 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
  5. 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.

 

Posted on: 7th September 2020

doi: https://doi.org/10.1242/prelights.24487

Read preprint (No Ratings Yet)




The authors respond to questions raised in the post

Joe Varberg and Sue Jaspersen shared

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?

J.V.: This is a great question – MYTH is sensitive enough to detect transient interactions, however there are likely multiple factors influencing whether specific interactions are detected or not in our study. The MYTH screen is being done in a heterologous system, so we recognize that the dynamics and regulation of the localization of the bait/preys could be different in this setting. Additionally, for Ima1, which does not have an ortholog the S. cerevisiae genome, the machinery control Ima1’s localization to the SPB could also be missing. Another consideration specifically for the putative SPB interactions we identified is that budding and fission yeast SPBs have different behaviors; whereas in budding yeast the SPBs remain permanently inserted in the nuclear envelope after duplication and insertion, fission yeast extrudes its SPBs at the completion of mitosis. All of this highlights two key points: first, that we aren’t visualizing where or when these interactions take place in the MYTH approach, and second, that the strength of interactions we detect in our screen reflect the protein behavior in the S. cerevisiae host and may not fully reflect their behavior in S. pombe. That being said, there is evidence that the strength of interactions reflects what’s happening in S. pombe. For example, Pom34 is known to complex with Cut11 at the NPC and is one of the stronger hits for Cut11 in MYTH, while the interaction with the SPB component Sad1 was weaker, potentially reflecting the transient localization of Cut11 to the SPB. Similarly, Nur1 forms a complex with Lem2 and was the second strongest hit for Lem2. Orthogonal approaches including co-immunoprecipitation, fluorescence cross-correlation spectroscopy and bimolecular fluorescence complementation are commonly used to further characterize the bait/prey interactions identified by MYTH screens. These approaches will be useful for follow up studies from our S. pombe MYTH hit to see when and where these proteins interact in fission yeast.

 

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.

J.V.: Indeed, a positive control bait would be ideal to test for expression and functionality of all prey in the library; however, this tool does not currently exist. One likely reason for this is that the residue that can be mutated to control the affinity between Nub and Cub (and allows for the Nub/Cub interaction to occur independently of an interaction between bait/prey) resides in the Nub fragment, which is present on the prey construct.

 

Can you speculate about reasons why some proteins are only functional as bait but not as prey and vice versa?

J.V.: First, while any protein can be used as a prey, bait proteins are required to be integral membrane proteins as soluble baits autoactivate. For the membrane proteins that we’ve seen only work in one direction, a likely culprit is protein topology in the membrane. Our prey library was designed using an approach that fuses Nub to the N-terminus of the prey protein. For most proteins in our library the topology has not been experimentally defined, and it’s possible that the N-terminus is not in the appropriate cellular compartment to interact with the C-termini of the MYTH bait proteins used in our screen. Another possible reason is that the bait and prey plasmids used in the MYTH assay are present in different copy number, with baits expressed from low-copy CEN plasmids to avoid artifacts of overexpression, and prey expressed from high-copy two micron plasmids. The bait and prey fusion proteins are also different sizes, and it’s possible that certain proteins can tolerate the smaller Nub tag but not the Cub/TF tag, with respect to protein folding and targeting to the membrane. 

 

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?

J.V.: It is possible that changes to protein structure or insertion/association with the membrane may occur due to introduction of the MYTH tags. This is one potential mechanism explaining why some of the bait constructs that we tried failed initial expression/functionality tests. In our previous experiments using MYTH in S. cerevisiae we found that MYTH fusion proteins (such as Ndc1/Cut11) were properly localized, however we did not directly test this for the fusion proteins used in the S. pombe screen.

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

Also in the cell biology category:

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020

 



List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.

 



List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria

 



List by Gautam Dey, Samantha Seah

1

Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.

 



List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20

 



List by Maiko Kitaoka, Madhuja Samaddar, Miguel V. Almeida, Sejal Davla, Jennifer Ann Black, Gautam Dey

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Preprint missing? Don't hesitate to let us know.

 



List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome

 



List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)

 



List by Madhuja Samaddar, Ramona Jühlen, Amanda Haage, Laura McCormick, Maiko Kitaoka

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019

 



List by Gautam Dey

Autophagy

Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.

 



List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.

 



List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.

 



List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019

 



List by Gautam Dey

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA

 



List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.

 



List by Gautam Dey, Amanda Haage
Close