Lights, Camera, Interaction: Studying Protein–Protein Interactions of the ER Protein Translocase in Living Cells

Abstract

Various landmark studies have revealed structures and functions of the Sec61/SecY complex in all domains of live demonstrating the conserved nature of this ancestral protein translocase. While the bacterial homolog of the Sec61 complex resides in the plasma membrane, the eukaryotic counterpart manages the transfer of precursor proteins into or across the membrane of the endoplasmic reticulum (ER). Sec61 complexes are accompanied by a set of dynamically recruited auxiliary proteins assisting the transport of certain precursor polypeptides. TRAP and Sec62/Sec63 are two auxiliary protein complexes in mammalian cells that have been characterized by structural and biochemical methods. Using these ER membrane protein complexes for our proof-of-concept study, we aimed to detect interactions of membrane proteins in living mammalian cells under physiological conditions. Bimolecular luminescence complementation and competition was used to demonstrate multiple protein–protein interactions of different topological layouts. In addition to the interaction of the soluble catalytic and regulatory subunits of the cytosolic protein kinase A, we detected interactions of ER membrane proteins that either belong to the same multimeric protein complex (intra-complex interactions: Sec61α–Sec61β, TRAPα–TRAPβ) or protein complexes in juxtaposition (inter-complex interactions: Sec61α–TRAPα, Sec61α–Sec63, and Sec61β–Sec63). In the process, we established further control elements like synthetic peptide complementation for expression profiling of fusion constructs and protease-mediated reporter degradation demonstrating the cytosolic localization of a reporter complementation. Ease of use and flexibility of the approach presented here will spur further research regarding the dynamics of protein–protein interactions in response to changing cellular conditions in living cells.

Proteomics Identifies Substrates and a Novel Component in hSnd2-Dependent ER Protein Targeting

“…We further verified the proximity between hSnd2 and TMEM109 under physiological conditions, i.e., in living cells using a split-luciferase system (NanoBiT) that we previously determined to be suitable for use with ER membrane proteins [ 70 , 71 ]. Briefly, this published work confirmed the well-known interactions of Sec61α with Sec61β, TRAPα, and Sec63, as well as between TRAPα and TRAPβ and Sec63 and Sec61β.…”

“…Briefly, this published work confirmed the well-known interactions of Sec61α with Sec61β, TRAPα, and Sec63, as well as between TRAPα and TRAPβ and Sec63 and Sec61β. Thus, it set the stage for addressing the putative interaction between hSnd2 and TMEM109 in living cells [ 71 ].…”

Signal Peptide Features Determining the Substrate Specificities of Targeting and Translocation Components in Human ER Protein Import

“…While the Sec61-complex mediates import of most precursor polypeptides into the ER, the Sec61-associated Sec62/Sec63 heterodimer supports ER protein import in a client-specific manner. Direct interaction between the Sec61 complex and Sec63 was demonstrated by co-immunoprecipitation as well as in living human cells (Tyedmers et al, 2000;Sicking et al, 2021b). Recently, four studies addressed the architecture of the posttranslationally acting translocon complex in yeast by cryoelectron microscopy (cryo-EM) (Itskanov and Park, 2019;Wu et al, 2019;Weng et al, 2021;Itskanov et al, 2021).…”

The Molecular Biodiversity of Protein Targeting and Protein Transport Related to the Endoplasmic Reticulum

“…While in yeast post-translationally targeted SPs show a lower hydrophobicity than the co-translational counterparts, post-translationally transported small secretory proteins of human cells show the opposite: an above-average hydrophobicity [ 17 , 81 ]. Irrespective of the organism, the transport of post-translational substrates requires the Sec61 complex to associate with the auxiliary membrane proteins Sec62/Sec63 that act as allosteric effectors supporting the opening of the translocation pore [ 82 , 83 , 84 , 85 , 86 , 87 , 88 ]. Other than SPs with a hydrophobicity deviant from the norm, human SPs with an above-average portion of the helix breaking residues glycine and proline require the presence of another auxiliary component, the translocon-associated protein (TRAP) complex [ 89 ].…”