Research Article

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

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Science  01 Feb 2019:
Vol. 363, Issue 6426, eaao0076
DOI: 10.1126/science.aao0076

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How rhomboid proteases act so quickly

How enzymes catalyze reactions in the viscous cell membrane is poorly understood. Kreutzberger et al. visualized single molecules of rhomboid intramembrane proteases diffusing in defined nanofabricated membranes and live cells (see the Perspective by Wolfe). They found that the rhomboid protein fold distorts surrounding lipids to reduce local membrane viscosity and enhance enzyme diffusion. The rate of catalysis in cells relies on rapid diffusion, revealing that rhomboid's diffusion has been boosted beyond the normal “speed limit” of the membrane, augmenting the search for substrates.

Science, this issue p. eaao0076; see also p. 453

Structured Abstract


Cell membranes are both a protective permeability barrier and a site where chemical reactions must be catalyzed to sustain life itself. How evolution adapted enzymes to this unusual environment is poorly understood. To shed mechanistic light on this gap, we focused on the rhomboid intramembrane serine protease superfamily that constitutes the most widely distributed membrane proteins across all life forms. These enzymes hydrolyze peptide bonds directly inside the membrane to liberate effector proteins in response to changing conditions. This deceptively simple reaction regulates a multitude of cellular events ranging from initiating developmental signaling in animals to dissolving adhesive contacts during parasite infection.


The membrane bilayer is a crowded and viscous environment through which rhomboid proteases must maneuver to find, bind, and cleave their transmembrane substrates. We rationalized that visualizing the mobility of individual rhomboid and substrate molecules in living cells (human and Drosophila) could provide a meaningful clue as to how these enzymes function in their environment. Armed with new bright and stable fluorophores and a sensitive microscope, we set out to track single molecules of diverse rhomboid proteins, various substrates, a series of canonical membrane proteins, and a phospholipid for comparison.


Tracking single molecules of a rhomboid endogenously expressed by human cells yielded a diffusion coefficient of ~0.8 μm2/s. This value exceeded the range for membrane proteins as measured by single-particle tracking. Unusually rapid diffusion proved to be a common feature of all 10 diverse rhomboid proteins that we studied across evolution and function. Indeed, rhomboid diffusion was comparable to the diffusion of small, single-pass transmembrane proteins, suggesting that rhomboid interacts differently with membrane lipids compared to other large, multispanning proteins.

To study this quality directly, we measured rhomboid diffusion relative to seven diverse proteins and a phospholipid standard in supported lipid bilayers of defined composition. Only rhomboid proteases diffused above the Saffman-Delbrück relation that defines the viscosity limit of diffusion in the membrane. Indeed, rhomboid proteins diffused at rates comparable to that of a 35-residue synthetic peptide, but how is this achieved?

An intriguing feature of the rhomboid fold is its irregular shape with thin hydrophobic segments that does not sit orderly in the membrane. Could this feature distort surrounding membrane lipids to reduce local viscosity and boost rhomboid diffusion? Indeed, we found that whereas all other membrane proteins diffused faster in thinner membranes, rhomboid diffusion slowed in thin membranes that matched its hydrophobic belt. Moreover, thickening membranes accelerated rhomboid diffusion. Finally, spectroscopic analysis revealed that a cytosolic appendage alters position of the rhomboid core in the membrane to accelerate diffusion further.

We lastly evaluated the contribution of rhomboid diffusion to the ultimate rate of proteolysis in living cells. Selectively slowing rhomboid diffusion reduced the rate of substrate proteolysis, whereas pharmacologically accelerating membrane diffusion greatly increased product formation. Rhomboid proteolysis thus relies on rapid diffusion through the membrane of living cells.


With their catalytic rate limited by diffusion, evolution sculpted the rhomboid fold to distort surrounding lipids, overcome the viscosity limit of the membrane, and accelerate its search for substrates. Our discovery reveals how evolution can boost the diffusion of enzymes in the crowded and viscous environment of the membrane.

This insight could have implications even beyond catalysis: Some rhomboid proteins that lost their catalytic residues still play important roles in membrane biology. Derlins, for example, facilitate endoplasmic reticulum (ER)–associated degradation of damaged proteins to safeguard the health of a cell. But without proteolytic activity, it has been difficult to rationalize their role. It is now tempting to speculate that Derlins disrupt local lipid interactions to help the Hrd1 channel translocate damaged proteins across the ER membrane.

Boosting protein diffusion through the membrane.

Hydrophobic mismatch with the membrane, repositioning by its cytosolic domain, and the irregular shape of rhomboid (red; top, right side) synergize to distort surrounding lipids (curved tails; top, right side) and boost its diffusion beyond the viscosity limit of the membrane (as shown in the graph, bottom left). Other membrane proteins (purple; top, left side) lacking these disruptive features (or subverting them in rhomboid, orange; bottom right) fit more regularly into the membrane and experience conventional diffusion.


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.

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