Much of the molecular motion in the cytoplasm is diffusive, which possibly limits the tempo of processes. We studied the dependence of protein mobility on protein surface properties and ionic strength. We used surface-modified fluorescent proteins (FPs) and determined their translational diffusion coefficients (D) in the cytoplasm of Escherichia coli, Lactococcus lactis and Haloferax volcanii. We find that in E. coli D depends on the net charge and its distribution over the protein, with positive proteins diffusing up to 100-fold slower than negative ones. This effect is weaker in L. lactis and Hfx. volcanii due to electrostatic screening. The decrease in mobility is probably caused by interaction of positive FPs with ribosomes as shown in in vivo diffusion measurements and confirmed in vitro with purified ribosomes. Ribosome surface properties may thus limit the composition of the cytoplasmic proteome. This finding lays bare a paradox in the functioning of prokaryotic (endo)symbionts.

The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to diffuse into the MCC/eisosomes, where a limited number of proteins are conditionally trapped at the (outer) edge of the compartment. Upon addition of substrate, the immobilized proteins diffuse away from the MCC/eisosomes, presumably after taking a different conformation in the substrate-bound state. Our data indicate that the mobile fraction of all integral plasma membrane proteins tested shows extremely slow Brownian diffusion through most of the PM. We also show that proteins with large cytoplasmic domains, such as Pma1 and synthetic chimera of Can1 and Lyp1, are excluded from the MCC/eisosomes. We hypothesize that the distinct localization patterns found for these integral membrane proteins in S. cerevisiae arises from a combination of slow lateral diffusion, steric exclusion, and conditional trapping in membrane compartments.

That diffusion is important for the proper functioning of cells is without question. The extent to which the diffusion coefficient is important is explored here for prokaryotic cells. We discuss the principles of diffusion focusing on diffusion-limited reactions, summarize the known values for diffusion coefficients in prokaryotes and in in vitro model systems, and explain a number of cases where diffusion coefficients are either limiting for reaction rates or necessary for the existence of phenomena. We suggest a number of areas that need further study including expanding the range of organism growth temperatures, direct measurements of diffusion limitation, expanding the range of cell sizes, diffusion limitation for membrane proteins, and taking into account cellular context when assessing the possibility of diffusion limitation.

SummaryWe measured translational diffusion of proteins in the cytoplasm and plasma membrane of the Grampositive bacterium Lactococcus lactis and probed the effect of osmotic upshift. For cells in standard growth medium the diffusion coefficients for cytosolic proteins (27 and 582 kDa) and 12-transmembrane helix membrane proteins are similar to those in Escherichia coli. The translational diffusion of GFP in L. lactis drops by two orders of magnitude when the medium osmolality is increased by ∼ 1.9 Osm, and the decrease in mobility is partly reversed in the presence of osmoprotectants. We find a large spread in diffusion coefficients over the full population of cells but a smaller spread if only sister cells are compared. While in general the diffusion coefficients we measure under normal osmotic conditions in L. lactis are similar to those reported in E. coli, the decrease in translational diffusion upon osmotic challenge in L. lactis is smaller than in E. coli. An even more striking difference is that in L. lactis the GFP diffusion coefficient drops much more rapidly with volume than in E. coli. We discuss these findings in the light of differences in turgor, cell volume, crowding and cytoplasmic structure of Gram-positive and Gram-negative bacteria.

Flagellar-driven motility grants unicellular organisms the ability to gather more food and avoid predators, but the energetic costs of construction and operation of flagella are considerable. Paths of flagellar evolution depend on the deviations between fitness gains and energy costs. Using structural data available for all three major flagellar types (bacterial, archaeal, and eukaryotic), flagellar construction costs were determined for Escherichia coli, Pyrococcus furiosus, and Chlamydomonas reinhardtii. Estimates of cell volumes, flagella numbers, and flagellum lengths from the literature yield flagellar costs for another ~200 species. The benefits of flagellar investment were analysed in terms of swimming speed, nutrient collection, and growth rate; showing, among other things, that the cost-effectiveness of bacterial and eukaryotic flagella follows a common trend. However, a comparison of whole-cell costs and flagellum costs across the Tree of Life reveals that only cells with larger cell volumes than the typical bacterium could evolve the more expensive eukaryotic flagellum. These findings provide insight into the unsolved evolutionary question of why the three domains of life each carry their own type of flagellum.