Abstract
SecA is a widely conserved ATPase that drives the secretion of proteins across the cell membrane via the SecYEG translocon, while the SRP system is a key player in the insertion of membrane proteins via SecYEG. How SecA gains access to substrate proteins in Bacillus subtilis cells and copes with an increase in substrate availability during biotechnologically desired, high-level expression of secreted proteins is poorly understood. Using single molecule tracking, we found that SecA localization closely mimics that of ribosomes, and its molecule dynamics change similarly to those of ribosomes after inhibition of transcription or translation. These data suggest that B. subtilis SecA associates with signal peptides as they are synthesized at the ribosome, similar to the SRP system. In agreement with this, SecA is a largely mobile cytosolic protein; only a subset is statically associated with the cell membrane, i.e., likely with the Sec translocon. SecA dynamics were considerably different during the late exponential, transition, and stationary growth phases, revealing that single molecule dynamics considerably alter during different genetic programs in cells. During overproduction of a secretory protein, AmyE, SecA showed the strongest changes during the transition phase, i.e., where general protein secretion is high. To investigate whether the overproduction of AmyE also has an influence on other proteins that interact with SecYEG, we analyzed the dynamics of SecDF, YidC, and FtsY with and without AmyE overproduction. SecDF and YidC did not reveal considerable differences in single molecule dynamics during overexpression, while the SRP component FtsY changed markedly in its behavior and became more statically engaged. These findings indicate that the SRP pathway becomes involved in protein secretion upon an overload of proteins carrying a signal sequence. Thus, our data reveal high plasticity of the SecA and SRP systems in dealing with different needs for protein secretion.