Calcium influx through plasma membrane calcium release-activated calcium (CRAC) channels, which are formed of hexamers of Orai1, is a potent trigger for many important biological processes, most notably in T cell-mediated immunity. Through a bioinformatics-led cell biological screen, we have identified Orai1 as a substrate for the rhomboid intramembrane protease RHBDL2. We show that RHBDL2 prevents stochastic calcium signaling in unstimulated cells through conformational surveillance and cleavage of inappropriately activated Orai1. A conserved disease-linked proline residue is responsible for RHBDL2's recognizing the active conformation of Orai1, which is required to sharpen switch-like signaling triggered by store-operated calcium entry. Loss of RHBDL2 control of CRAC channel activity causes severe dysregulation of downstream CRAC channel effectors, including transcription factor activation, inflammatory cytokine expression, and T cell activation. We propose that this surveillance function may represent an ancient activity of rhomboid proteases in degrading unwanted signaling proteins.

• Keywords
• CRAC channel, Orai1, RHBDL2, T cell, calcium, rhomboid protease, signalling, transmembrane,
• MeSH
• Lymphocyte Activation MeSH
• Cell Membrane metabolism   MeSH
• Drosophila melanogaster MeSH
• Ion Channel Gating MeSH
• HEK293 Cells MeSH
• Protein Conformation MeSH
• Humans MeSH
• Membrane Proteins metabolism   MeSH
• Mutation MeSH
• Peptide Hydrolases chemistry   MeSH
• ORAI1 Protein chemistry   MeSH
• Serine Endopeptidases metabolism   MeSH
• Signal Transduction MeSH
• Stochastic Processes MeSH
• Calcium metabolism   MeSH
• Calcium Signaling physiology   MeSH
• Calcium Channels chemistry   MeSH
• Protein Binding MeSH
• Computational Biology MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Proteins MeSH
• ORAI1 protein, human MeSH Browser
• Orai1 protein, mouse MeSH Browser
• Peptide Hydrolases MeSH
• ORAI1 Protein MeSH
• RHBDL2 protein, human MeSH Browser
• RHBDL2 protein, mouse MeSH Browser
• Serine Endopeptidases MeSH
• Calcium MeSH
• Calcium Channels MeSH

Magnesium homeostasis is essential for life and depends on magnesium transporters, whose activity and ion selectivity need to be tightly controlled. Rhomboid intramembrane proteases pervade the prokaryotic kingdom, but their functions are largely elusive. Using proteomics, we find that Bacillus subtilis rhomboid protease YqgP interacts with the membrane-bound ATP-dependent processive metalloprotease FtsH and cleaves MgtE, the major high-affinity magnesium transporter in B. subtilis. MgtE cleavage by YqgP is potentiated in conditions of low magnesium and high manganese or zinc, thereby protecting B. subtilis from Mn2+ /Zn2+ toxicity. The N-terminal cytosolic domain of YqgP binds Mn2+ and Zn2+ ions and facilitates MgtE cleavage. Independently of its intrinsic protease activity, YqgP acts as a substrate adaptor for FtsH, a function that is necessary for degradation of MgtE. YqgP thus unites protease and pseudoprotease function, hinting at the evolutionary origin of rhomboid pseudoproteases such as Derlins that are intimately involved in eukaryotic ER-associated degradation (ERAD). Conceptually, the YqgP-FtsH system we describe here is analogous to a primordial form of "ERAD" in bacteria and exemplifies an ancestral function of rhomboid-superfamily proteins.

• Keywords
• ER-associated degradation, intramembrane protease, membrane transporter, proteostasis, rhomboid,
• MeSH
• ATPases Associated with Diverse Cellular Activities metabolism   MeSH
• Bacillus subtilis growth & development   metabolism   MeSH
• Bacterial Proteins metabolism   MeSH
• Endopeptidases metabolism   MeSH
• Membrane Proteins metabolism   MeSH
• Proteomics methods   MeSH
• Gene Expression Regulation, Bacterial MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ATPases Associated with Diverse Cellular Activities MeSH
• Bacterial Proteins MeSH
• Endopeptidases MeSH
• Membrane Proteins MeSH

Many membrane proteins are thought to function as dimers or higher oligomers, but measuring membrane protein oligomerization in lipid membranes is particularly challenging. Förster resonance energy transfer (FRET) and fluorescence cross-correlation spectroscopy are noninvasive, optical methods of choice that have been applied to the analysis of dimerization of single-spanning membrane proteins. However, the effects inherent to such two-dimensional systems, such as the excluded volume of polytopic transmembrane proteins, proximity FRET, and rotational diffusion of fluorophore dipoles, complicate interpretation of FRET data and have not been typically accounted for. Here, using FRET and fluorescence cross-correlation spectroscopy, we introduce a method to measure surface protein density and to estimate the apparent Förster radius, and we use Monte Carlo simulations of the FRET data to account for the proximity FRET effect occurring in confined two-dimensional environments. We then use FRET to analyze the dimerization of human rhomboid protease RHBDL2 in giant plasma membrane vesicles. We find no evidence for stable oligomers of RHBDL2 in giant plasma membrane vesicles of human cells even at concentrations that highly exceed endogenous expression levels. This indicates that the rhomboid transmembrane core is intrinsically monomeric. Our findings will find use in the application of FRET and fluorescence correlation spectroscopy for the analysis of oligomerization of transmembrane proteins in cell-derived lipid membranes.

• MeSH
• Cell Membrane metabolism   MeSH
• Dimerization MeSH
• Humans MeSH
• Membrane Lipids metabolism   MeSH
• Membrane Proteins * metabolism   MeSH
• Protein Multimerization MeSH
• Fluorescence Resonance Energy Transfer * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Lipids MeSH
• Membrane Proteins * MeSH