Abstract
Biological membranes have been proposed to contain microdomains of a specific lipid composition, in which distinct groups of proteins are clustered. Flotillin-like proteins are conserved between pro—and eukaryotes, play an important function in several eukaryotic and bacterial cells, and define in vertebrates a type of so-called detergent-resistant microdomains. Using STED microscopy, we show that two bacterial flotillins, FloA and FloT, form defined assemblies with an average diameter of 85 to 110 nm in the model bacterium Bacillus subtilis. Interestingly, flotillin microdomains are of similar size in eukaryotic cells. The soluble domains of FloA form higher order oligomers of up to several hundred kDa in vitro, showing that like eukaryotic flotillins, bacterial assemblies are based in part on their ability to self-oligomerize. However, B. subtilis paralogs show significantly different diffusion rates, and consequently do not colocalize into a common microdomain. Dual colour time lapse experiments of flotillins together with other detergent-resistant proteins in bacteria show that proteins colocalize for no longer than a few hundred milliseconds, and do not move together. Our data reveal that the bacterial membrane contains defined-sized protein domains rather than functional microdomains dependent on flotillins. Based on their distinct dynamics, FloA and FloT confer spatially distinguishable activities, but do not serve as molecular scaffolds.
Highlights
In spite of many decades of research on membrane proteins, the true arrangement of proteins and their dynamics within the lipid bilayer are still poorly defined
Using super resolution fluorescence microscopy, we show that several Bacillus subtilis membrane proteins cluster into structures of 60 to 110 nm, verifying the existence of defined-size protein microdomains
Biochemical co-isolation of specific membrane proteins and flotillins, a family of proteins highly conserved between eukaryotic and bacterial cells, suggested that common “functional” microdomains exist, containing so-called “detergent-resistant” membrane proteins, that are centered by flotillins
Summary
In spite of many decades of research on membrane proteins, the true arrangement of proteins and their dynamics within the lipid bilayer are still poorly defined. Flotillin/reggie proteins (reggies/flotillins, prohibitins, podocins, stomatins) are an evolutionarily conserved class of proteins found across all organisms [9]. They are considered as regulators of membrane protein trafficking [10] and as common constituents of DRMs in eukaryotic cells. Flotillins are involved in membrane-trafficking, in signal transduction, and cytoskeletal rearrangement [3, 10]. They are discussed as scaffolding proteins and as couplers of membrane-proteins with the actin cytoskeleton [12, 13]. During axon growth in neuronal cells, flotillins are suggested to induce membrane microdomain formation at the growth cone [14, 15] and to recruit specific proteins to the elongating axon [16]
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