Site-directed fluorescence labeling reveals a revised N-terminal membrane topology and functional periplasmic residues in the Escherichia coli cell division protein FtsK [Cell Biology]

July 7th, 2014 by Berezuk, A. M., Goodyear, M., Khursigara, C. M.

In Escherichia coli, FtsK is a large integral membrane protein that coordinates chromosome segregation and cell division. The N-terminal domain of FtsK (FtsKN) is essential for division, while the C-terminus (FtsKC) is a well-characterized DNA translocase. Although the function of FtsKN is unknown, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation. This may occur through modulation of protein interactions between FtsKN and other division proteins in both the periplasm and cytoplasm; thus a clear understanding of how FtsKN is positioned in the membrane is required to characterize these interactions. The membrane topology of FtsKN was initially determined using site-directed reporter fusions, however questions regarding this topology persist. Here, we report a revised membrane topology generated by site-directed fluorescence labeling. The revised topology confirms the presence of four transmembrane segments and reveals a newly identified periplasmic loop between the third and fourth transmembrane domains. Within this loop, four residues were identified that, when mutated, resulted in the appearance of cellular voids. High-resolution transmission electron microscopy of these voids showed asymmetric division of the cytoplasm, and inhibition of cell wall ingrowth and outer membrane invagination. This uncoupling reveals a novel role for FtsK in linking cell envelope septation events, and yields further evidence for FtsK as a critical checkpoint of cell division. The revised topology of FtsKN also provides an important platform for future studies on essential interactions required for this process.
  • Posted in Journal of Biological Chemistry, Publications
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