Abstract
Crescentin is a bacterial filament-forming protein that exhibits domain organization features found in metazoan intermediate filament (IF) proteins. Structure-function studies of eukaryotic IFs have been hindered by a lack of simple genetic systems and easily quantifiable phenotypes. Here we exploit the characteristic localization of the crescentin structure along the inner curvature of Caulobacter crescentus cells and the loss of cell curvature associated with impaired crescentin function to analyze the importance of the domain organization of crescentin. By combining biochemistry and ultrastructural analysis in vitro with cellular localization and functional studies, we show that crescentin requires its distinctive domain organization, and furthermore that different structural elements have distinct structural and functional contributions. The head domain can be functionally subdivided into two subdomains; the first (amino-terminal) is required for function but not assembly, while the second is necessary for structure assembly. The rod domain is similarly required for structure assembly, and the linker L1 appears important to prevent runaway assembly into nonfunctional aggregates. The data also suggest that the stutter and the tail domain have critical functional roles in stabilizing crescentin structures against disassembly by monovalent cations in the cytoplasm. This study suggests that the IF-like behavior of crescentin is a consequence of its domain organization, implying that the IF protein layout is an adaptable cytoskeletal motif, much like the actin and tubulin folds, that is broadly exploited for various functions throughout life from bacteria to humans. © 2011 Wiley-Liss, Inc.
Highlights
Bacteria have a complex subcellular architecture, a notion that has only recently become apparent thanks to advances in microscopy techniques
It is clear that bacteria have exploited the flexibility of the core tubulin and actin folds to create proteins with properties tailored to specific functions as diverse as cell shape maintenance, plasmid segregation, and cell division [Cabeen and Jacobs-Wagner, 2010]
Cells were imaged at room temperature ($ 22C) on a Nikon E1000 microscope fitted with 100Â differential interference contrast or phase-contrast objectives and a Hamamatsu Orca-ER LCD camera, or a Nikon E80i microscope fitted with similar objectives and a Hamamatsu Orca-IIER LCD camera
Summary
Bacteria have a complex subcellular architecture, a notion that has only recently become apparent thanks to advances in microscopy techniques. AlfA was used as a starting point to search for more bacterial actin homologs too divergent in sequence to be identified using MreB as a reference sequence. This approach uncovered as many as 35 additional families of bacterial actin homologs, all with signature actin motifs [Derman et al, 2009]. It is clear that bacteria have exploited the flexibility of the core tubulin and actin folds to create proteins with properties tailored to specific functions as diverse as cell shape maintenance, plasmid segregation, and cell division [Cabeen and Jacobs-Wagner, 2010]
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