AbstractThere are two major protein translocases for protein export across the bacterial cytoplasmic membrane - the general secretory (Sec) system and the twin-arginine translocation (Tat) system. Both translocases can also insert membrane proteins. The Sec machinery inserts multiple transmembrane domains into the cytoplasmic membrane by a co-translational mechanism, whereas, the Tat machinery translocates fully folded proteins across the membrane and is only known to integrate proteins that carry a single transmembrane helix.
The Rieske protein is a membrane-bound iron-sulphur protein found in bacterial electron transport chains. In most bacteria, assembly of Rieske is Tat-dependent, as iron-sulphur cofactor insertion occurs in the cytoplasm, and the protein usually contains a single transmembrane helix anchor. However, in Actinobacteria, this protein comprises 3 transmembrane domains prior to the cofactor-containing domain, causing a biosynthetic insertion problem. Previous work has shown that the Streptomyces coelicolor Rieske protein is dual targeted to the cytoplasmic membrane since it requires both the Sec and Tat export pathways for correct insertion.
The aim of this study was to investigate this dual-targeting mechanism and to understand the specific features of Rieske that facilitate its release by the Sec machinery and its subsequent recognition by the Tat machinery for complete insertion into the membrane. Development of reporter assays allowed assessment of the interaction of the transmembrane domains of Rieske with either the Sec or Tat pathway. It was discovered that topological determinants, like hydrophobicity and the positive-inside rule, are crucial to the Sec machinery releasing the third transmembrane domain. In comparison, the Tat machinery requires at least seven residues in the cytoplasmic loop preceding the Tat motif for efficient recognition of the membrane-tethered substrate. This work has elucidated the mechanism by which assembly of the Actinobacterial Rieske protein is co-ordinated.
|Date of Award||2016|
|Sponsors||Medical Research Council, The Biochemical Society & Microbiology Society|
|Supervisor||Tracy Palmer (Supervisor)|