Translocation of the precursor of α‐amylase into Bacillus subtilis membrane vesicles

Abstract

Bacilli vigorously secrete proteins into the extracellular environment, and are therefore used in industry for the bulk production of enzymes such as proteinases and amylases. Studies on the mechanism of protein translocation in these Gram-positive bacteria have been hampered by the lack of an in vitro system. To establish such a system for Bacillus subtilis, everted membranes were isolated from a strain deficient in the alkaline and neutral protease. Translocation-competent membrane vesicles were obtained only when a broad range proteinase-inhibitor cocktail was used during membrane isolation. This method efficiently prevented proteolysis of SecY, one of the core integral membrane components of the preprotein translocase. Translocation of the urea-denatured precursor of the Bacillus licheniformis A-amylase, preAmyL, and B. subtilis alkaline phosphatase, prePhoB, into the B. subtilis membrane vesicles require the B. subtilis SecA protein and are driven by ATP hydrolysis and the proton-motive force. These studies establish an authentic in vitro translocation system for protein secretion in B. subtilis.

Re-annotating the Mycoplasma pneumoniae genome sequence: adding value, function and reading frames

“…The YvaL homology has also been reported by Bellgard and Gojobori (38). YvaL has in the meantime been experimentally verified to be a SecG homolog (39).…”

Detergent-independent in vitro activity of a truncated Bacillus signal peptidase

“…The most likely explanation of why pre-A2-AmyL preparations are not completely processed is that some molecules of this precursor become incompetent for processing. In vitro this can be due to (micro)aggregation of the precursor, which leads to translocation and processing incompetence (van Wely et al, 1998). In fact, this would explain why the use of different precursor preparations resulted in different maximum levels of processing.…”

Identification of Two Interaction Sites in SecY that Are Important for the Functional Interaction with SecA

“…Translocation buffer consists of 50 mM Hepes/NaOH (pH 7.0), 20 mM KCl, 5 mM MgCl 2 , 0.1 mg/ml bovine serum albumin (BSA), 5 mM DTT. SecA was iodinated 41 and IMVs were isolated 23 as described.…”