The rate of folding dictates substrate secretion by the Escherichia coli hemolysin type 1 secretion system.
Secretion of the Escherichia coli toxin hemolysin A (HlyA) is catalyzed by the membrane protein complex HlyB-HlyD-TolC and requires a secretion sequence located within the last 60 amino acids of HlyA. The Hly translocator complex exports a variety of passenger proteins when fused N-terminal to this secretion sequence. However, not all fusions are secreted efficiently. Here, we demonstrate that the maltose binding protein (MalE) lacking its natural export signal and fused to the HlyA secretion signal is poorly secreted by the Hly system. We anticipated that folding kinetics might be limiting secretion, and we therefore introduced the "folding" mutation Y283D. Indeed this mutant fusion protein was secreted at a much higher level. This level was further enhanced by the introduction of a second MalE folding mutation (V8G or A276G). Secretion did not require the molecular chaperone SecB. Folding analysis revealed that all mutations reduced the refolding rate of the substrate, whereas the unfolding rate was unaffected. Thus, the efficiency of secretion by the Hly system is dictated by the folding rate of the substrate. Moreover, we demonstrate that fusion proteins defective in export can be engineered for secretion while still retaining function.
Institute of Biochemistry, Heinrich Heine University Duesseldorf, Universitaetsstrasse 1, 40225 Duesseldorf, Germany.
Patrick J
PJ
Bakkes
Stefan
S
Jenewein
Sander H J
SH
Smits
I Barry
IB
Holland
Lutz
L
Schmitt
D019943:Amino Acid Substitution; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D058947:Bacterial Secretion Systems / Q000502:physiology*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D033902:Periplasmic Binding Proteins / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C065231:HlyD protein, E coli; C036507:Hlya protein, E coli; C050837:Hlyb protein, Bacteria; C461619:MalE protein, E coli; D026901:Membrane Transport Proteins; D046912:Multiprotein Complexes; D033902:Periplasmic Binding Proteins; C073126:tolC protein, E coli