title: An internal hydrophobic helical domain of Bacillus subtilis enolase is essential but not sufficient as a non-cleavable signal for its secretion.
abstract: Many cytoplasmic proteins without a cleavable signal peptide, including enolase, are secreted during the stationary phase in Bacillus subtilis but the molecular mechanism is not yet clear. We previously identified a highly conserved embedded membrane domain in an internal hydrophobic α-helix of enolase that plays an important role in its secretion. In this study, we examined the role of the helix in more detail for the secretion of enolase. Altering this helix by mutations showed that many mutated forms in this domain were not secreted, some of which were not stable as a soluble form in the cytoplasm. On the other hand, mutations on the flanking regions of the helix or the conserved basic residues showed no deleterious effect. Bacillus enolase with the proper hydrophobic helical domain was also exported extracellularly in Escherichia coli, indicating that the requirement of the helix for the secretion of enolase is conserved in these species. GFP fusions with enolase regions showed that the hydrophobic helix domain itself was not sufficient to serve as a functional secretion signal; a minimal length of N-terminus 140 amino acids was required to mediate the secretion of the fused reporter GFP. We conclude that the internal hydrophobic helix of enolase is essential but is not sufficient as a signal for secretion; the intact long N-terminus including the hydrophobic helix domain is required to serve as a non-cleavable signal for the secretion of Bacillus enolase.
authors.affiliation: Department of Biology, and Center of Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, United States.
authors.forename: Chun-Kai
authors.initials: CK
authors.lastname: Yang
authors.affiliation: Department of Biology, and Center of Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, United States.
authors.forename: Xiao-Zhou
authors.initials: XZ
authors.lastname: Zhang
authors.affiliation: Department of Biology, and Center of Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, United States.
authors.forename: Chung-Dar
authors.initials: CD
authors.lastname: Lu
authors.affiliation: Department of Biology, and Center of Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, United States. Electronic address: biopct@gsu.edu.
authors.forename: Phang C
authors.initials: PC
authors.lastname: Tai
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010751:Phosphopyruvate Hydratase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D021382:Protein Sorting Signals; D010751:Phosphopyruvate Hydratase
keywords: Enolase; Hydrophobic domain; Secretion