Quantitative genome-scale analysis of protein localization in an asymmetric bacterium

Abstract

Despite the importance of subcellular localization for cellular activities, the lack of high-throughput, high-resolution imaging and quantitation methodologies has limited genomic localization analysis to a small number of archival studies focused on Cterminal fluorescent protein fusions. Here, we develop a highthroughput pipeline for generating, imaging, and quantitating fluorescent protein fusions that we use for the quantitative genomic assessment of the distributions of both N-and C-terminal fluorescent protein fusions. We identify nearly 300 localized Caulobacter crescentus proteins, up to 10-fold more than were previously characterized. The localized proteins tend to be involved in spatially or temporally dynamic processes and proteins that function together and often localize together as well. The distributions of the localized proteins were quantitated by using our recently described projected system of internal coordinates from interpolated contours (PSICIC) image analysis toolkit, leading to the identification of cellular regions that are over-or under-enriched in localized proteins and of potential differences in the mechanisms that target proteins to different subcellular destinations. The Caulobacter localizome data thus represent a resource for studying both global properties of protein localization and specific protein functions, whereas the localization analysis pipeline is a methodological resource that can be readily applied to other systems.bacteria ͉ Caulobacter ͉ genomics ͉ quantitative image analysis ͉ high-throughput imaging T he emergence of high-throughput methods to assay the levels of cellular components such as mRNAs and proteins has enabled the study of biological processes at a systems level, enhancing the understanding of both individual proteins and functional modules. Meanwhile, the subcellular localizations of proteins that mediate such cellular processes as growth, division, and motility indicate that many important aspects of cell biology require precise spatial organization. The significance of this localization holds true in both eukaryotes and prokaryotes. Large-scale efforts have cataloged the localizations of Cterminal GFP fusions to most of the proteins of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Escherichia coli (1-3). However, the utility of these libraries has been limited by the laborious nature of generating, imaging, and scoring fluorescent fusions, thereby restricting their reanalysis under different conditions and reconstruction as N-terminal fusions or fusions to other fluorescent proteins. Furthermore, protein localization has classically been analyzed via qualitative descriptors that limit their utility for quantitative systems-level characterizations.Despite their small size and lack of internal compartmentalization, bacterial cells are highly organized (4). Relatively small genomes and experimental tractability thus make bacteria powerful systems for studying universal cell biological processes such as cell division, cell cycle progression, a...

Caulobacter PopZ forms an intrinsically disordered hub in organizing bacterial cell poles

“…Some have hypothesized that intrinsically disordered hub proteins are advantageous in complex systems because they simplify multicomponent networks by allowing many binding partners to be organized around a single hub (15,14). Dozens of different proteins are localized specifically to Caulobacter cell poles (2). In this light, the role of PopZ in organizing such a complex network in bacteria is consistent with the functions of other intrinsically disordered hub proteins.…”

“…In rod-shaped bacteria, the cell poles are also sites for the assembly of complex structures. Electron microscopy has shown that the cytoplasm at the cell poles in Caulobacter is visibly distinct from other areas of the cytoplasm (1), and a genome-wide screen identified more than 80 different proteins that are localized to these locations (2). Although some of these proteins are known to require the presence of an upstream recruitment factor, a fully ordered system of polar assembly has not been established.…”

Focal targeting by human β-defensin 2 disrupts localized virulence factor assembly sites in Enterococcus faecalis

“…1) because they were most abundant within the log-phase population under study (Experimental Procedures). For quantitative image analysis, we used Projected System of Internal Coordinates from Interpolated Contours (PSICIC), a program originally developed for elongated cells such as E. coli and Caulobacter crescentus (31,32). We extended the program to record the fluorescence intensity at the perimeter of ovococcal and diplococcal E. faecalis cells, enabling the rapid quantification of localized fluorescence measurements at the membrane of the bacteria.…”

Protein localization and dynamics within a bacterial organelle

“…Until recently, no molecular probes for the study of the stalk ultrastructure and biogenesis were known. Conducting a cytological survey of 75% of the predicted translation products of Caulobacter, Werner et al uncovered candidate proteins that appear associated with the stalk (6). The stalk is a cylindrical protrusion of all envelope layers (inner membrane, periplasm, outer membrane, and S-layer) at the old cell pole and encloses cytoplasmic material that is free of chromosomal DNA, ribosomes, and most cytoplasmic proteins (7,8).…”