Entropy-driven translocation of disordered proteins through the Gram-positive bacterial cell wall

Cells across all kingdoms of life actively partition molecules between discrete cellular compartments. In Gram-positive bacteria, a thick and highly cross-linked peptidoglycan cell wall separates the bacterial membrane from the extracellular space, imposing a barrier that must be crossed by proteins whose functions require that they be exposed on the bacterial cell surface 1,2 . Some surface-exposed proteins, such as the Listeria monocytogenes actin nucleation-promoting factor ActA 3 , remain associated with the bacterial membrane yet somehow thread through tens of nanometers of dense, cross-linked cell wall to expose their N-terminus on the outer surface 4,5 . Here, we show that entropy can drive the translocation of disordered transmembrane proteins through the Gram-positive cell wall. We develop a physical model predicting that the entropic constraint imposed by a thin periplasm is sufficient to drive translocation of an intrinsically disordered protein like ActA across a porous barrier similar to the cell wall. Consistent with this scenario, we demonstrate experimentally that translocation depends on both the dimensions of the cell envelope and the length of the disordered protein, and that translocation is reversible. We also show that disordered regions from eukaryotic nuclear pore complex proteins are capable of entropy-driven translocation through Gram-positive cell walls. These observations suggest that entropic forces alone, rather than chaperones or chemical energy, are sufficient to drive translocation of certain Gram-positive surface proteins for exposure on the outer surface of the cell wall.