An In Vitro Assay to Study Targeting of Membrane Proteins to the Inner Nuclear Membrane

Abstract

Newly synthesized membrane proteins are inserted into the endoplasmic reticulum (ER) from where they are constantly sorted to various cellular compartments. To analyze and visualize sorting of membrane proteins to the inner nuclear membrane (INM), we developed a trap-release system that uncouples membrane integration into the ER from transport. This assay allows the simultaneous release of a large pool of an INM-destined membrane protein from the ER and microscopy-based monitoring of targeting to the INM. The use of semi-permeabilized HeLa cells further enables the identification and characterization of essential requirements of the targeting process. This protocol provides a detailed description of reporter construction, in vitro reconstitution, and visualization of trafficking.

Efficient protein targeting to the inner nuclear membrane requires Atlastin-dependent maintenance of ER topology

“…Addition of TEV protease to the semi-permeabilized cells allows for a fast cleavage of the tandem RFPs, thereby enabling NPC passage of the reporters. Targeting to the INM is then reconstituted by supplementing HeLa cell lysate and an energy regenerating system (ATP, GTP, creatine phosphate and creatine kinase; later referred to as energy) ( Ungricht et al, 2016 ). Accumulation of the GFP-tagged reporter proteins at the NE over time can be followed by time-lapse microscopy allowing for a quantitative description of the INM targeting process.…”

“…In vitro INM targeting reactions were performed as described previously ( Ungricht et al, 2016 ). In brief, reporter cells were semi-permeabilized with permeabilization buffer (PB: 20 mM Hepes pH 7.5, 110 mM potassium acetate, 5 mM magnesium acetate, 0.5 mM EGTA, 250 mM sucrose) containing 0.0025% digitonin for 10 min at 4°C, followed by three washes with PB for 2, 5, and 10 min, respectively.…”

“…Quantification of INM targeting levels was done as described ( Ungricht et al, 2016 ). An ImageJ plugin was used to detect nuclear contours of DAPI-stained nuclei and the ER border by thresholding on the eGFP channel.…”

A role for Nup153 in nuclear assembly reveals differential requirements for targeting of nuclear envelope constituents

“…It is of note that the architecture of the endoplasmic reticulum, and particularly the role of Atlastin, is established to be important for INM protein diffusion through the ER and subsequent targeting to the NE at interphase (Pawar et al, 2017). There has also been considerable focus on how proteins target to the INM during ongoing recruitment of newly-synthesized INM proteins at interphase (Boni et al, 2015;King et al, 2006;Lokareddy et al, 2015;Meinema et al, 2011;Ohba et al, 2004;Rempel et al, 2020;Ungricht et al, 2016). These studies point toward pathways for INM protein passage through the NPC, after which interactions at the nuclear periphery may aid in retention.…”