PrsA LIPOPROTEIN AND POSTTRANSLOCATIONAL FOLDING OF SECRETORY PROTEINS IN BACILLUS SUBTILIS
Year: 2004
Type: article
Abstract: Protein folding is crucial for proteins in order to gain their correct three-dimensional conformation and become functionally active.In most cases this folding process needs the assistance of chaperones and folding catalysts.In this thesis the posttranslocational folding of secretory proteins in Bacillus subtilis was studied, namely the folding of exoproteins once they have been translocated across the cytoplasmic membrane into the membrane-cell wall interface and need to fold correctly in this environment.A special focus was on the role of PrsA lipoprotein in this posttranslocational folding process.The aim was to further characterize the PrsA protein with a structure-function analysis and to study its role as a limiting factor in secretion.PrsA protein was essential for viability of cells; its depletion resulted in abnormal filamentous growth eventually leading to cell lysis.PrsA was an abundant protein on the outer surface of the cell membrane.The number of PrsA molecules per cell was estimated to be about 20 000 molecules, yet only a few hundred molecules per cell were enough to support normal growth.PrsA exhibited an enzymatic peptidyl prolyl cis/trans isomerase (PPIase) activity in vitro.The middle domain, which is homologous to other known parvulin-type PPIases, was alone sufficient for this enzymatic activity.Yet all three domains (the N-terminal, PPIase and C-terminal domain) were essential for the function of PrsA in vivo in terms of secretion of α-amylase and cell viability.An insertion mutagenesis further characterized the importance of the N-terminal domain since insertions in this part of the protein totally inactivated or reduced the PrsA activity unlike insertions in the PPIase and C-terminal domains in which the insertions were mostly tolerated.Saturation of the secretion machinery sets limits on the translocation and secretion and thus engineering of components of the secretion machinery is necessary for enhanced function in biotechnical applications.Saturation of the secretion machinery was studied using AmyQ α-amylase as a model protein and the role of PrsA as a limiting factor for secretion of AmyQ was studied in this context.The capacity of secretion apparatus was determined to be 10 fg/h/cell at the late logarithmic phase of growth meaning a secretion of 30 AmyQ molecules/second.PrsA deficiency did not decrease the capacity of protein translocation confirming that PrsA was not involved in the translocation event itself.Instead, the rate of signal processing was found to be a limiting factor for the translocation of AmyQ.PrsA was a limiting factor for AmyQ secretion in conditions where the AmyQ was overproduced and there was a low level of PrsA but unexpectedly, also in reversed conditions when there was an excess of PrsA compared to the level of AmyQ.Efficient posttranslocational folding is necessary when heterologous proteins are produced and secreted into the culture medium in B. subtilis.Therefore, the effect of PrsA protein and two other factors affecting the late stages of protein folding, namely the negative net charge of the cell wall and the HtrA-type quality control proteases in the membrane-cell wall matrix were studied in this context.A set of eleven proteins with biotechnological interest was used in these experiments.The secretion of four of these model proteins was dependent on PrsA and overproduction of PrsA enhanced secretion of two of them, α-amylase of B. stearothermophilus (4fold) and pneumolysin (1.5-fold).Increasing the net negative charge of the cell wall by mutating the dlt operon responsible for the D-alanylation of teichoic acids enhanced the secretion of pneumolysin about 1.5-fold.Decreasing the level of HtrA-type quality control proteases caused harmful effects on growth and did not enhance secretion.The pertussis toxin subunit S1 was found to be a substrate for HtrA-type proteases and its secretion was dependent on these proteases.Results indicate that when components involved in the posttranslocational folding are engineered, secretion of some heterologous proteins is enhanced.
Author: Marika Vitikainen
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2163019360