The rough endoplasmic reticulum (ER) of nucleated human cells has crucial functions in protein biogenesis, calcium (Ca²⁺) homeostasis, and signal transduction. Among the roughly one hundred components, which are involved in protein import and protein folding or assembly, two components stand out: The Sec61 complex and BiP. The Sec61 complex in the ER membrane represents the major entry point for precursor polypeptides into the membrane or lumen of the ER and provides a conduit for Ca²⁺ ions from the ER lumen to the cytosol. The second component, the Hsp70-type molecular chaperone immunoglobulin heavy chain binding protein, short BiP, plays central roles in protein folding and assembly (hence its name), protein import, cellular Ca²⁺ homeostasis, and various intracellular signal transduction pathways. For the purpose of this review, we focus on these two components, their relevant allosteric effectors and on the question of how their respective functional cycles are linked in order to reconcile the apparently contradictory features of the ER membrane, selective permeability for precursor polypeptides, and impermeability for Ca²⁺. The key issues are that the Sec61 complex exists in two conformations: An open and a closed state that are in a dynamic equilibrium with each other, and that BiP contributes to its gating in both directions in cooperation with different co-chaperones. While the open Sec61 complex forms an aqueous polypeptide-conducting- and transiently Ca²⁺-permeable channel, the closed complex is impermeable even to Ca²⁺. Therefore, we discuss the human hereditary and tumor diseases that are linked to Sec61 channel gating, termed Sec61-channelopathies, as disturbances of selective polypeptide-impermeability and/or aberrant Ca²⁺-permeability.

Looking at the variety of the thousands of different polypeptides that have been focused on in the research on the endoplasmic reticulum from the last five decades taught us one humble lesson: no one size fits all. Cells use an impressive array of components to enable the safe transport of protein cargo from the cytosolic ribosomes to the endoplasmic reticulum. Safety during the transit is warranted by the interplay of cytosolic chaperones, membrane receptors, and protein translocases that together form functional networks and serve as protein targeting and translocation routes. While two targeting routes to the endoplasmic reticulum, SRP (signal recognition particle) and GET (guided entry of tail-anchored proteins), prefer targeting determinants at the N- and C-terminus of the cargo polypeptide, respectively, the recently discovered SND (SRP-independent) route seems to preferentially cater for cargos with non-generic targeting signals that are less hydrophobic or more distant from the termini. With an emphasis on targeting routes and protein translocases, we will discuss those functional networks that drive efficient protein topogenesis and shed light on their redundant and dynamic nature in health and disease.

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.