Abstract
Many bacterial proteins require specific subcellular localization for function. How Escherichia coli proteins localize at one pole, however, is still not understood. Here, we show that the DnaK (HSP70) chaperone controls unipolar localization of the Shigella IpaC type III secretion substrate. While preventing the formation of lethal IpaC aggregates, DnaK promoted the incorporation of IpaC into large and dynamic complexes (LDCs) restricted at the bacterial pole through nucleoid occlusion. Unlike stable polymers and aggregates, LDCs show dynamic behavior indicating that nucleoid occlusion also applies to complexes formed through transient interactions. Fluorescence recovery after photobleaching analysis shows DnaK-IpaC exchanges between opposite poles and DnaKJE-mediated incorporation of immature substrates in LDCs. These findings reveal a key role for LDCs as reservoirs of functional DnaK-substrates that can be rapidly mobilized for secretion triggered upon bacterial contact with host cells.
Highlights
Many bacterial proteins require specific subcellular localization for function
A genome wide screen of proteins fused to green fluorescent protein (GFP) implicated the DnaK chaperone in IcsA polarization, but how this chaperone controls IcsA polar localization remains unclear[12]
To identify IpaC polarization determinants, we performed pull-down experiments with a hexahistidine-tagged construct of IpaC lacking the U region fused to GFP (CiΔU-His) that localizes to the pole and is produced at high levels (Fig. 1a, b)
Summary
Many bacterial proteins require specific subcellular localization for function. How Escherichia coli proteins localize at one pole, is still not understood. While preventing the formation of lethal IpaC aggregates, DnaK promoted the incorporation of IpaC into large and dynamic complexes (LDCs) restricted at the bacterial pole through nucleoid occlusion. Mechanisms describing “functional” cytoplasmic protein localization at bacterial poles remain controversial, since amorphous aggregates are excluded from the nucleoid and accumulate at the poles or forming septa. In the “diffusion-capture” mechanism, proteins are targeted through interactions with a polar localization determinant This mechanism has been proposed for proteins involved in cell division and chromosome segregation in E. coli, cell cycle control in Bacillus subtilis and Caulobacter crescentus (reviewed in ref.[2]). The Shigella type III translocon component IpaC localizes to one bacterial pole prior to cell contact and determines polar type III secretion during host cell invasion by Shigella[11].
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