Rhomboid distorts lipids to break the viscosity-imposed speed limit of membrane diffusion

Abstract

Enzymes that cut proteins inside membranes regulate diverse cellular events, including cell signaling, homeostasis, and host-pathogen interactions. Adaptations that enable catalysis in this exceptional environment are poorly understood. We visualized single molecules of multiple rhomboid intramembrane proteases and unrelated proteins in living cells (human and Drosophila) and planar lipid bilayers. Notably, only rhomboid proteins were able to diffuse above the Saffman-Delbrück viscosity limit of the membrane. Hydrophobic mismatch with the irregularly shaped rhomboid fold distorted surrounding lipids and propelled rhomboid diffusion. The rate of substrate processing in living cells scaled with rhomboid diffusivity. Thus, intramembrane proteolysis is naturally diffusion-limited, but cells mitigate this constraint by using the rhomboid fold to overcome the “speed limit” of membrane diffusion.

A predicted plastid rhomboid protease affects phosphatidic acid metabolism in Arabidopsis thaliana

“…Even though rhomboids do not recognize a consensus sequence, their substrate recognition mechanism offers surprising specificity for their transmembrane helix substrates. Moreover, rhomboids scan for their substrate moving through membranes at biologically relevant rates due to the nature of their interaction with the membrane (Kreutzberger et al ., ). Unfortunately, the lack of a concrete cleavage consensus site poses a challenge when searching for an endogenous substrate and ultimately a molecular mechanism to explain the observed rbl10 lipid phenotypes.…”

Practical Considerations in Particle and Object Tracking and Analysis

“…Taking these considerations together, single‐particle tracking can be a powerful method to generate novel biological insights. For example, Urban and colleagues recently used SPT‐TIRF microscopy to show surprisingly unique dynamic behavior of membrane‐bound rhomboid proteins in several cell types (Kreutzberger, Ji, Aaron, Mihaljević, & Urban, ). This example illustrates many of the aspects discussed above.…”

“…Figure (adapted with permission from Kreutzberger et al., ) shows HEK293T cells transfected with N‐Halo‐tagged rhomboid‐like 2 (RHBDL2) proteins, and labeled with Janelia Fluor 549 dye. In Figure A, cells were first imaged in normal imaging medium; Figure B shows the same field of view after treatment with Mg 2+ and ionomycin.…”

“…This method of trajectory visualization can also be used to identify areas of relative high and low particle diffusion. Results adapted with permission from Kreutzberger et al (2019).…”

“…Subsequently, particles were detected via thresh-old application, localized via Gaussian fitting, and positions liked via nearest-neighbor analysis to form trajectories using the Mosaic particle tracking plugin for ImageJ (Sbalzarini & Koumoutsakos, 2005), one of a number of freely available tracking algorithms . Figure 19 (adapted with permission from Kreutzberger et al, 2019) shows HEK293T cells transfected with N-Halotagged rhomboid-like 2 (RHBDL2) proteins, and labeled with Janelia Fluor 549 dye. In Figure 19A, cells were first imaged in normal imaging medium; Figure 19B shows the same field of view after treatment with Mg 2+ and ionomycin.…”