Deciphering Protein Evolution

One of the enduring questions in biology is how eukaryotic cells arose from prokaryotic ancestors at least 2 billion years ago. Besides differences in genome organization, eukaryotic animals, plants, and fungi possess a much higher degree of cellular compartmentation in the form of membrane bound organelles than their distant bacterial and Archaean cousins. But how did such a plethora of cellular domains, each with a discrete role in metabolism, evolve? To the extent that science proves anything, it answered the question for two eukaryotic organelles a long time ago. Mitochondria and chloroplasts evolved from endosymbiotic associations between an ancestral host cell and smaller prokaryotic partners. In the case of chloroplasts, the symbiont was a photosynthetic cyanobacterium; for mitochondria, most likely it was ana-proteobacterium. The cytoplasm of eukaryotic cells is like chicken soup-it's chock full of organelles suspended like chunks of assorted vegetables and noodles in cytosolic broth. The broth also contains filaments of various dimensions that collectively comprise the cell's cytoskeleton. Like the bones of a large animal, the cytoskeleton provides a structural framework lending shape to cells and against which enzymatic 'muscles' work to elicit movement. That's how amoebae migrate, algae swim, stem cells divide, and cytoplasm streams relentlessly up, down, and across plant cells. While the cytoskeleton is as much a hallmark of eukaryoticity as any mitochondrion or chloroplast, the origin of its filaments in deep time is more mysterious. Biologists assumed that genes for cytoskeletal proteins arose from prokaryotic precursors, but evidence in favor of the hypothesis was scarce, until recently. Tubulin First on Stage Microtubules comprise one component of the cytoskeleton responsible for a variety of movements including mitosis and meiosis. The 25 nm tubes consist of dimerica- and b-tubulin subunits that share about 40 percent sequence homology. Another form,y-tubulin, functions in microtubule formation. But where did microtubules come from? It now appears that tubulins share a common ancestor with a protein called FtsZ, a key player in bacterial cell division.1 FtsZ is also present in plants, where it functions in chloroplast division,2 and a similar protein associates with mitochondria, at least in one alga.3 FtsZ polymerizes into filaments in the test tube in a process dependent on GTP. The same nucleotide is required for tubulin assembly into microtubules.1 Tubulins and FtsZ are clearly related, judging from similarities in three-dimensional structure. And although the proteins share only about 15 percent amino acid sequence identity overall, they're much more similar at the local level, particularly at the domain responsible for binding and cleaving GTP.4,5 Actin Into the Fold Like the tubulins, actin-another essential component of the eukaryotic cytoskeleton-is a globular protein that binds nucleotide, in this case ATP. As actin monomers polymerize into 6-nm-wide microfilaments consisting of two helically wound protofilaments, the ATP, situated in a deep enzymatic cleft between two halves of the protein, hydrolyzes to ADP and inorganic phosphate. It turns out that actin shares its ATPase domain with a family of proteins including hexokinase, the enzymatic kick starter of glycolysis, and several bacterial proteins. One of them is called MreB, a protein essential for generating or maintaining the rod shape of many bacteria. By examining structural similarities between eukaryotic actin and MreB from Thermotoga maritima, a research team at the Medical Research Council in Cambridge, England recently concluded that the two proteins are more closely related to each other than to other members of the family and undoubtedly share a common ancestor.6 The group showed that the three-dimensional shapes of actin and MreB are so similar they can be superimposed. The analogy with tubulin/FtsZ goes even further. Both proteins share considerable amino acid homology at several key sequences surrounding the ATP binding site, again situated deep in a cleft between two halves of the folded polypeptide chain. Under the right conditions, MreB polymerizes into protofilaments that pair up lengthwise. The protein subunits are spaced about the same distance apart along the filaments as in polymeric actin, but MreB double filaments aren't nearly as helical. The similarity between MreB and actin doesn't stop at structure and sequence. In a paper published earlier in 2001, a research group led by Jeffrey Errington at the University of Oxford, U.K. visualized MreB in the rod shaped cells of Bacillus subtilis using fluorescence and electron microscopy.7 MreB forms filamentous bands that encircle the cell in low helices, like reinforcing hoops. In an essay accompanying the Cambridge group's article, Duke University cell biologist Harold Erickson calculated that each band contains 10 protofilaments.8 When Errington's team genetically deprived cells of functional MreB, they became spherical. A search of genome databases showed that MreB is present in bacteria with nonspherical shapes, including rods. It's absent in spherical cocci. In other words, MreB has a cytoskeletal function. "I think it is quite convincing that MreB is the actin progenitor," says Erickson. "A key step, still unknown, going from bacteria to vertebrates is to develop a mechanism to make the double-helical actin filament from the single MreB protofilament structure." More Acts to Follow The story doesn't end with MreB; there's more to find out. Scientists want to know if MreB is also present in eukaryotes-associated with mitochondria and chloroplasts-as is FtsZ. According to Katherine Osteryoung, a plant biologist at Michigan State University in East Lansing who identified two FtsZ genes in the mustard plant Arabidopsis,2 "there's no obvious indication of MreB in plants that I've found or am aware of." Actin normally functions along with the motor enzyme myosin to produce cellular motion, while microtubules utilize two other motor families called dynein and kinesin related proteins. Researchers now wonder whether MreB and FtsZ work in conjunction with bacterial motors. According to Erickson, "none have been turned up in genetic screens for cell division (or other activities), and none have been identified by sequence gazing. My bet is that kinesin and myosin evolved in eukaryotes, after the evolution of microtubules and eukaryotic actin filaments." Still, Osteryoung is pleased with the latest results: "To someone interested in these issues, establishment of the prokaryotic origins of two major eukaryotic cytoskeletal proteins is enormously satisfying. I look forward to the day when evolutionary intermediates... from MreB to actin and FtsZ to tubulin, perhaps awaiting discovery in some obscure and primitive eukaryote, will more fully reveal the evolutionary steps by which key components of the eukaryotic cytoskeleton acquired their present-day structures and functions." Barry A. Palevitz (palevitz@dogwood.botany.uga.edu) is a contributing editor for The Scientist. References 1. H.P. Erickson, "FtsZ, a tubulin homologue in prokaryotic cell division," Trends in Cell Biology, 7:362-7, 1997. 2. K.W. Osteryoung, "Organelle fission: Crossing the evolutionary divide," Plant Physiology, 123:1213-6, 2000. 3. P.L. Beech et al., "Mitochondrial FtsZ in a chromophyte alga," Science, 287:1276-9, 2000. 4. E. Nogales et al., "Structure of the alpha-beta tubulin dimer by electron crystallography," Nature, 391:199-203, 1998. 5. J. Lowe, L.A. Amos, "Crystal structure of the bacterial cell-division protein FtsZ," Nature, 391:203-6, 1998. 6. F. Van den Ent et al., "Prokaryotic origin of the actin cytoskeleton," Nature, 413:39-44, Sept. 2, 2001. 7. L.J.F. Jones et al., "Control of cell shape in bacteria: helical, actin-like filaments in Bacillus subtilis," Cell, 104:913-22, 2001. 8. H.P. Erickson, "Evolution in bacteria," Nature, 413:30, Sept. 6, 2001.

Deciphering Protein Evolution

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