A TAle of Two Pathways: Tail-Anchored Protein Insertion at the Endoplasmic Reticulum

Guna, Alina; Hazu, Masami; Tomaleri, Giovani Pinton; Voorhees, R.M.

doi:10.1101/cshperspect.a041252

Cited by 11 publications

(15 citation statements)

References 126 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The proximity of the TMD to the stop codon necessitates that TAs be targeted and inserted into the membrane post-translationally. Though found in all cellular membranes, the majority of TAs are targeted to the ER using two parallel pathways: the Guided Entry of Tail-anchored protein (GET) and ER membrane complex (EMC) pathways (Stefanovic & Hegde, 2007; Schuldiner et al, 2008; Guna et al, 2018; Guna et al, 2022a).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens

Guna

Page

Replogle

et al. 2023

Preprint

View full text Add to dashboard Cite

The ability to map genetic interactions has been essential for determining gene function and defining biological pathways. Therefore, a system to readily perform genome-wide genetic modifier screens in human cells is a powerful platform for dissecting complex processes in mammalian cells, where redundancy and adaptation commonly mask the phenotype of a single genetic perturbation. Here, we report a CRISPR interference (CRISPRi) based platform, compatible with Fluorescence Activated Cell Sorting (FACS)-based reporter screens, that can be used to query epistatic relationships at scale. This is enabled by a flexible dual-sgRNA library design that allows for the simultaneous delivery and selection of a fixed sgRNA and a second randomized guide, comprised of a genome-wide library, with a single transduction. As a proof of principle, we apply our approach to study the pathways that mediate tail-anchored (TA) protein insertion at the endoplasmic reticulum (ER). We show that this dual-guide library approach can be successfully coupled with FACS-based reporter screening, to identify genetic epistasis and thereby place TA biogenesis factors in their respective parallel pathways. We demonstrate that this dual-guide approach is both more sensitive and specific than traditional growth screening approaches, and is ideally suited for dissecting the complex interplay between factors in human cells.

show abstract

Section: Resultsmentioning

confidence: 99%

“…Cell lines expressing GFP1-10 in the ER lumen were generated as previously described (Inglis et al, 2020; Guna et al, 2022b). CRISPRi K562 cells were infected with lenti virus containing CalR(GFP1-10)-KDEL and sorted with a Sony Cell Sorter (SH800S) as single clones into 96-well plates.…”

Section: Methodsmentioning

confidence: 99%

A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens

Guna

Page

Replogle

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…The human genome harbors a multitude of complete membrane proteins, with the formation of membrane protein compartments occurring predominantly at the ER in most cells. In the case of multichannel proteins, the SRP sequesters a nascent polypeptide in the cytosol to facilitate transport of the ribosomal nascent chain complex to the ER membrane 45 . Once within the ER, the TMD must be embedded into the lipid bilayer, a process comprising two steps: first, transfer of the hydrophobic TMD from the aqueous cytosol to the membrane‐spanning topology within the lipid bilayer, followed by the transmembrane movement of the associated soluble domains into the ER lumen.…”

Section: The Functions Of Emcmentioning

confidence: 99%

“…In the case of multichannel proteins, the SRP sequesters a nascent polypeptide in the cytosol to facilitate transport of the ribosomal nascent chain complex to the ER membrane. 45 Once within the ER, the TMD must be embedded into the lipid bilayer, a process comprising two steps: first, transfer of the hydrophobic TMD from the aqueous cytosol to the membrane-spanning topology within the lipid bilayer, followed by the transmembrane movement of the associated soluble domains into the ER lumen. The latter step, being energetically unfavorable, is typically catalyzed by membrane protein insertion enzymes within the cell.…”

Section: Emc As An Insertasementioning

confidence: 99%

ER membrane complex (EMC): Structure, functions, and roles in diseases

Zhu,

Zhang

2024

The FASEB Journal

View full text Add to dashboard Cite

The endoplasmic reticulum (ER) is the largest membrane system in eukaryotic cells and is the primary site for the biosynthesis of lipids and carbohydrates, as well as for the folding, assembly, modification, and transport of secreted and integrated membrane proteins. The ER membrane complex (EMC) on the ER membrane is an ER multiprotein complex that affects the quality control of membrane proteins, which is abundant and widely preserved. Its disruption has been found to affect a wide range of processes, including protein and lipid synthesis, organelle communication, endoplasmic reticulum stress, and viral maturation, and may lead to neurodevelopmental disorders and cancer. Therefore, EMC has attracted the attention of many scholars and become a hot field. In this paper, we summarized the main contributions of the research of EMC in the past nearly 15 years, and reviewed the structure and function of EMC as well as its related diseases. We hope this review will promote further progress of research on EMC.

show abstract

“…One important family of membrane proteins that rely on their TMD and its flanking residues for recognition, targeting, and insertion are tail-anchored proteins (TAs) (Kutay et al, 1993; Chio et al, 2017; Hegde and Keenan, 2011; Guna et al, 2022a). TAs are characterized by a single C-terminal TMD followed by a short soluble domain of up to 30-40 amino acids.…”

Section: Introductionmentioning

confidence: 99%

A selectivity filter in the EMC limits protein mislocalization to the ER

Pleiner

Hazu

Tomaleri

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

Tail anchored proteins (TAs) play essential roles at both the ER and mitochondria, and their accurate localization is critical to proteostasis. Biophysical similarities lead to mistargeting of mitochondrial TAs to the ER, where they are delivered to the ER membrane protein complex (EMC). We showed that the EMC directly contributes to sorting fidelity of mitochondrial TAs and multipass substrates that contain positively charged soluble domains. Leveraging an improved structural model of the human EMC, we used mutagenesis and site-specific crosslinking to map the path of a TA from its cytosolic capture by methionine-rich loops to its membrane insertion through a hydrophilic vestibule. Positively charged residues at the entrance to the vestibule function as a selectivity filter that uses charge-repulsion to reject mitochondrial TAs. Substrate discrimination by the EMC provides a biochemical explanation for one role of charge in TA sorting and protects compartment identity by limiting protein misinsertion.

show abstract

A TAle of Two Pathways: Tail-Anchored Protein Insertion at the Endoplasmic Reticulum

Cited by 11 publications

References 126 publications

A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens

A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens

ER membrane complex (EMC): Structure, functions, and roles in diseases

A selectivity filter in the EMC limits protein mislocalization to the ER

Contact Info

Product

Resources

About