An engineered transcriptional reporter of protein localization identifies regulators of mitochondrial and ER membrane protein trafficking in high-throughput screens

Robert Coukos, David Yao, Mateo I. Sanchez, Eric T. Strand, Jonathan S. Weissman, Michael C. Bassik, Alice Y. Ting

Preprint posted on 12 April 2021

Article now published in eLife at

Keep it simple! HiLITR is a novel fluorescent reporter converting complex phenotypes into a simple readout suitable for pooled genetic screens.

Selected by Lorenzo Lafranchi



Gene perturbation screens have been widely used for identifying the molecular players involved in diverse cellular processes. Pooled screens are particularly powerful, because they are easy to assemble and can be used to probe libraries of more than 105 unique elements. One limitation of this approach is that a perturbation of the cellular process of interest needs to be linked to a simple readout, such as a change in survival, proliferation, or increased/decreased expression of a reporter fluorophore. In the latter case, cells of interest can be enriched using fluorescence-activated cell sorting (FACS). More complex phenotypes, such as changes in the subcellular localization of a protein of interest, requires a high-content microscopy readout and need to be screened using an arrayed format. Arrayed screens are usually nosier, limited to a smaller number of perturbations (in the range of 103-104) and technically more difficult. In an effort to combine the strengths of these two screening approaches, Coukos and colleagues develop a molecular reporter that converts a complex phenotype such as protein mislocalization to a simple fluorescent readout.


Key findings

HiLITR (High-throughput Localization Indicator with Transcriptional Readout)

HiLITR, the fluorescent reporter presented in this study, includes two components: a GFP-coupled TEV protease and a transcription factor (TF) with a linker region including the protease cleave sequence (TEVcs). Both constructs can be independently targeted to specific subcellular localizations and if they are in close proximity, the activity of the protease releases the transcription factor which can translocate to the nucleus and drive expression of a chosen reporter gene. To better control the activity of the protease, the authors included a photosensory LOV domain, which sterically masks the TEVcs until it is displaced upon exposure to blue light (Figure 1). After showcasing the efficacy of their system by targeting the two components either to the same or two different subcellular localizations (i.e. the outer mitochondrial membrane (OMM), the endoplasmic reticulum membrane (ERM) and/or the cytosol), the authors use HiLITR to discover novel factors controlling the trafficking and membrane insertion of mitochondrial and ER proteins.


Figure 1 (A) Schematics depicting the HiLITR reporter and (B) fluorescence images of HiLITR in HeLa cells. Only when the protease and the transcription factor are localizing to the same subcellular structure, the transcription factor can be released and drive expression of a reporter gene (i.e. mCherry). Figure is modified from Figure 1 of the preprint, which was made available under a CC-BY 4.0 International license.


CRISPRi screens to probe pathways of ER and mitochondrial proteins insertion

Membrane insertion is dictated by the presence of N-terminal (signal-anchor), C-terminal (tail-anchor), internal or multi-pass transmembrane domains in a specific protein. Several distinct, but tightly intertwined pathways regulate the co-translational and post-translational insertion of membrane proteins at the OMM and ERM. Recent studies helped elucidate the mechanisms involved in regulating protein trafficking to mitochondria and ER membranes, but many nodes of these pathways still await to be defined. In particular, the mechanisms controlling insertion of tail-anchored mitochondrial proteins remain elusive. To fill this gap, Coukos and colleagues used HiLITR to performed a genome-wide CRISPRi screen, in which they combine a tail-anchored mitochondrial protease with a signal-anchored mitochondrial transcription factor. Using this design, any genetic perturbation that would affect trafficking and insertion of tail-anchored OMM proteins would cause a reduction in the expression of the fluorescent reporter. Based on the results of the genome-wide screen, the authors designed a smaller library comprising sgRNAs targeting 586 genes-of-interest that was then used in three further screens, which relied on varied localizations of the HiLITR components. This approach allowed the authors to clarify the role of the different hits in mitochondrial- or ER-specific trafficking pathways, but also elegantly exclude false-positive hits that would intrinsically emerge from the initial genome-wide screen.

Reassured by the presence of genes known to be involve in mitochondrial and ER protein trafficking in their hits list, the authors turned their interest to novel genes that might specifically influence the targeting of mitochondrial tail-anchored proteins. Interestingly, only one protein was able to fulfill the most stringent criteria used to cross-evaluate the results of the three independent “validation” screens: SAE1. SAE1 is a member of the SAE complex, which is the only E1 SUMO-activating enzyme present in mammalian cells, and it is strictly required to enable protein SUMOylation. SUMOylation is a widespread post-translational modification, which can alter the interactome or subcellular localization of the target protein, as well as regulate its stability. Following up on this, the authors show that downregulation of SAE1 affects overall stability and mitochondrial localization of three tail-anchored proteins. Importantly, localization and stability of non-tail-anchored mitochondrial proteins are unaffected by SAE1 depletion.

Finally, in one of the three screens performed using the sgRNA sublibrary (the “ER screen”), the protease is present at both the OMM and the ERM, allowing the identification of novel regulators of ER tail-anchored proteins. By examining hits that decreased HiLITR activation in this screen, the authors identified several components of the ER membrane complex (EMC). This result agrees with the role of EMC in promoting tail-anchored proteins insertion in the ER membrane. Differently from other EMC subunits, EMC10 depletion led to increased HiLITR signal, suggesting that this specific subunit plays a role in regulating and/or antagonizing the activity of the ER membrane complex. To corroborate this observation, the authors further demonstrate that depletion of EMC10 results in the mislocalization of the ER-localized protease and the stabilization of the EMC client protein SQS.


What I like about this work and future directions

The HiLITR reporter presented in this manuscript is an elegant way of linking complex phenotypes such as protein localization and trafficking to a simple and robust fluorescent readout, which enables the collection of cells displaying a phenotype-of-interest by FACS. This modular reporter system will therefore facilitate the study of a variety of cellular processes involving changes in protein localization using the power of pooled screening approaches.


Questions to the authors

Would the cytoplasmic expression of both HiLITR components induce expression of the reporter gene?

You mentioned that several chaperones involved in the deposition of tail-anchored proteins are SUMOylated. Did you investigate whether depletion of these chaperones recapitulates the phenotypes observed upon SAE1 depletion? Were these chaperones identified as hits in the genome-wide screen?

Are MAVS, SYNJ2BP and FIS1 SUMOylated?

Tags: crispr, fluorescent reporter, genetic screen, transmembrane proteins

Posted on: 31 May 2021


Read preprint (No Ratings Yet)

Author's response

Robert Coukos and Alice Y. Ting shared

Would the cytoplasmic expression of both HiLITR components induce expression of the reporter gene?

If the HiLITR transcription factor were not robustly excluded from the nucleus, it would drive reporter expression independent of interaction with a cytoplasmic protease construct. However, if the transcription factor were contained only in the cytoplasm, reporter expression would be a function of the expression levels of both HiLITR components as well as the duration of light stimulation. Decreased expression levels and light stimulation times could likely provide for a context in which activation would not occur, while increased expression and light stimulation could likely provide for activation even if the TF and protease were not canonical binding partners. We suspect that either circumstance could be arranged with some tuning of the aforementioned parameters.


You mentioned that several chaperones involved in the deposition of tail-anchored proteins are SUMOylated. Did you investigate whether depletion of these chaperones recapitulates the phenotypes observed upon SAE1 depletion? Were these chaperones identified as hits in the genome-wide screen?

We did not directly follow up on the chaperones. However, several were included in the sublibrary screens which were informed by the whole-genome screen. STIP1 depletion decreases HiLITR activation in all three screens. However, STIP1 is not specific to TA proteins, so this result is expected. In contrast to STIP1, the ubiquilins showed neutral effects in all three screens, which is likely a consequence of functional redundancy with each other when only one is knocked down. We speculate that the effect of SAE1 depletion, if indeed mediated through the chaperones, is a result of one of the following possibilities. First, SUMOylation may tune the specificity of chaperones with respect to TA and non-TA proteins. Second, reduced SUMOylation may tax protein trafficking throughout the cell, and TA proteins, which are not locally translated, may be more severely impacted by moderate defects in trafficking.


Are MAVS, SYNJ2BP and FIS1 SUMOylated?

There is some data available on proteome-wide SUMOylation, and these proteins do not appear to be SUMOylated. This data represents a steady-state, and it doesn’t exclude the possibility that these proteins are transiently SUMOylated during maturation and trafficking to the outer mitochondrial membrane. However, we have no reason to believe this is the case, and we suspect that the effect of SAE1 depletion is more indirect.

Have your say

Your email address will not be published.

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:

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.


List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.


List by Nadja Hümpfer et al.


The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!


List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.


List by Alex Eve

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 Dey Lab, Samantha Seah


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 et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.


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 et al.

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 Dey Lab


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 Dey Lab


This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.


List by Sandra Franco Iborra

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 Dey Lab, Amanda Haage

Also in the molecular biology category: