Protein localization and asymmetry in the bacterial cell

Cell, Vol. 73, 841-855,

June 4, 1993, Copyright

0 1993 by Cell Press

Review

Protein Localization and Asymmetry in the Bacterial Cell Lucille Shapiro Department of Developmental Biology Beckman Center Stanford University School of Medicine Stanford, California 94305

types occurs upon an asymmetric cell division or by communication between homologousor heterologouscellsfollowing division (Horvitz and Herskowitz, 1992). Although these mechanisms are not mutually exclusive, in this review I examine cell-intrinsic differentiation potential, while Kaiser and Losick (1993) consider the role of cell-cell interactions in bacterial behavior and development.

Introduction Localization Cellular architecture can change as the cell cycle progresses and as differentiated cell types are established. These programmed changes can occur in free-living organisms, such as single-celled bacteria and fungi, or as components of highly structured groups of cells in embryos or tissues of organisms of all phyla. Minimally, bacterial cells follow a complex cascade of signals to replicate their chromosomes, effect cell division, and respond to environmental cues. Recent evidence has revealed that the bacterial cell-even in its most basic, workaday existence-is organized into functional domains at specific sites in the cell. Some of these domains, such as those containing the cell division machinery, assemble and disassemble during the cell cycle. Others, such as those containing the chemotaxis machinery, are located at the poles of the cell. How cellular signals dictate the deposition of protein complexes to these functional domains in the cell is a major problem in understanding the cellular and developmental biology of all cells. There are several conditions that result in cellular asymmetry. Some unicellular organisms are inherently asymmetric: the site of division in the bacterial cell or the site of bud formation in the yeast Saccharomyces cerevisiae may be influenced by cellular markers that are laid down at the previous division site. Independent of or in addition to this inherent asymmetry, communication with other cells or changes in the nutrient or chemical environment impose restrictionson cellular organization. For more specialized behavior, some bacteria, such as Caulobacter, differentiate polar structures that allow them to forage for food for only a portion of their cell cycle. Other bacteria, such as Bacillus or Myxococcus, differentiate into spores to protect their genome from certain doom when living conditions become intolerable. In the case of the myxobacteria, aggregation into clusters of cells with distinct, and sometimes exotic, morphological features precedes spore formation (Kaiser and Losick, 1993 [this issue of Cc//j). Changes in cellular organization are also observed in bacterial pathogens during invasion of host cells (Bliska et al., 1993 [this issue of Cc/d), in blue-green algae heterocyst formation (Haselkorn and Buikema, 1992), and in bacterial endosymbiants providing essential biochemical functions to a plant cell (Long and Staskawicz, 1993 [this issue of Cc/i)). In each of these cases, cellular rearrangements are brought about by changes in gene expression, protein distribution, or both. The generation of differentiated cell

of Biochemical

Functions

The acceptance of the eukaryotic cell as a department store of goods and services, with asymmetrically distributed domains, is based on the extensive characterization of its specialized intracellular membranous organelles. Ensuing discoveries that proteins and protein complexes, which are not associated with these intracellular organelles, are localized to specific regions of the plasma membrane provided an additional dimension to our understanding of cellular architecture. For example, mechanisms are being investigated for the sequestering of different proteins to specific domainsof the plasma membrane of polarized epithelial cells (such as the sequestering of Na+/K+ATPase to the basal-lateral domain; Rodriguez-Boulan and Nelson, 1989), the localization of the yeast a receptor to the tip of the projection formed in mating a cells (Jackson et al., 1991) and the localization of chemoreceptors in mammalian neutrophils to the tip of the pseudopodial extensions (Walter et al., 1980; Weinbaum et al., 1980). It has come as something of a surprise to find that the bacterial cell, devoid of intracellular organelles and small enough to rely on diffusion to get most of its business done, also localizes protein complexes to specific regions of the cell. The most extensively characterized protein complexes that are localized in the bacterial cell are the cell division apparatus (Bi and Lutkenhaus, 1991) and the chemosensory apparatus (Alley et al., 1992, 1993; Maddock and Shapiro, 1993). The localization of each of these bacterial complexes is compared in the following sections with the parallel establishment of chemoreceptors in mammalian neutrophils, of bud sites in yeast, and the asymmetric distribution of mating receptors in yeast. The Cell Division Apparatus A spatially restricted event most accessible to study in unicellular organisms is cell division. The selection of a site for cell division and the mechanics of the division process are controlled by an intricate genetic network in both the bacterial cell and the eukaryotic yeast cell. Although the actual players are different, many facets of the process are analogous in yeast and bacteria. In most bacteriaincluding Escherichia coli and Bacillus subtilis-and the fission yeast Schizosaccharomyces pombe, the cell division plane is equatorial. In contrast, highly asymmetric division sites are formed during the initiation of sporulation

Cell a42

initiates FtsZ ring fornation at a specific site on the cell

4 GTP

GTP

recndbnent of other cell division proteins

GTP

I 1

c

Fts.1, FtsA

/ i

F&Q, F&L

1

contraction of F&Z ring

GTP GTP GTP

activation of FtsZ GTPase

Figure 1. The Role of FtsZ in E. coli Cell Division The model is derived primarily from the work of Bi and Lutkenhaus (1991). The indicated GTP binding and GTPase activities of FtsZ are from Mukherjee et al. (1993). RayChaudhuri and Park (1992), and de Boer et al. (1992). At this time, the existence of a guanine nucleotide exchange factor and a protein that activates the FtsZ GTPase is consistent with other G protein systems, but has not been demonstrated. Prior to the initiation of cell division, FtsZ is uniformly dispersed throughout the cytoplasm, most likely in the GDP-bound form. The division process initiates with the accumulation of FtsZ at the equator of the cell. It has been suggested that the exchange of bound GTP for GDP accompanies polymerization of a FtsZ-GTP ring on the cytoplasmic face of the inner membrane. Other division proteins-Ftsl, FtsA, FtsQ, and FtsL-are recruited to the division plane. The FtsZ ring appears to contract and then depolymerize when division is completed. Contraction, depolymerization, or both would be accompanied by the activation of the FtsZ GTPase activity.

in 6. subtilis (reviewed in Losick and Stragier, 1992) during heterocyst formation in the blue-green algae Anabaena (Haselkorn and Buikema, 1992; Wolk, 1991) and in the budding yeast S. cerevisiae (reviewed in Horvitz and Herskowitz, 1992). The factors that control division site selection in bacteria are not known. However, it has been argued that topological features of the cell surface influence site selection (de Boer et al., 1989) and that the cell might measure the distance between the cell pole and the division plane by virtue of its accommodation of the newly replicated nucleoids (Nanninga et al., 1991; Woldringh et al., 1991). The Bacterial Cell An early event in the establishment of the division plane in E. coli is the localization of a GTP-binding protein, FtsZ, to the inner surface of the cytoplasmic membrane at the equator of the cell (Bi and Lutkenhaus, 1991). For much of the cell cycle, the FtsZ protein is randomly distributed

throughout the cytoplasm. The initiation of ring formation appears to occur only after nuclear segregation is complete. In rapidly growing E. coli, there are approximately 20,000 monomers of FtsZ per cell (Bi and Lutkenhaus, 1991). These monomers are apparently sequestered to the division site to participate in the assembly of the division machinery (Figure 1). As the division process proceeds, the FtsZ ring, which is attached to the leading edge of the invagination of the cytoplasmic membrane, decreases in diameter. Once division is completed, the FtsZ ring disassembles. It has been demonstrated that the binding of FtsZ to guanine nucleotides is critical to its function in the cell division process (Mukherjee et al., 1993; RayChaudhuri and Park, 1992; de Boer et al., 1992). The FtsZ protein binds GTP using the same sequence motif as is used by tubulin (Gaelen and Haley, 1979). Additional proteins, such as a GDP-GTP exchange factor and a protein that activates the GTPase activity of FtsZ, may participate in septal site selection (Figure 1). An attractive hypothesis, borne out by examination of the FtsZ polymerization process in vitro, is that exchange of bound GDP by GTP is the cue that initiates ring formation at the equator of the cell and activates multimer formation in a manner analogous to polymerization of the GTPbound form of tubulin (Snyder and McIntosh, 1976). A strain containing a defective f&Z allele in the presence of a wild-type tiszgene exhibits a dominant negative phenotype, as expected for a protein whose function is to polymerize subunits (Mukherjee et al., 1993). The folding or assembly of FtsZ monomers may be mediated by the DnaK chaperone, as DnaK is found in association with FtsZ and dnaK deletion mutants are defective in the division process. The endogenous GTPase activity of the E. coli FtsZ protein is concentration dependent in vitro, suggesting that this activity is triggered at a certain stage of polymerization in vivo. This has led to the speculation that GTPase activation contributes to the contraction of the FtsZ ring, to its depolymerization, or both (Mukherjee et al., 1993; de Boer et al., 1992; Figure 1). The FtsZ protein originally identified and localized in E. coli is highly conserved among eubacteria- including B. subtilis (Beall and Lutkenhaus, 1991), Rhizobium meliloti (Margolin et al., 1991) and Caulobacter crescentus (Y. Brun and L. S., unpublished data)-and has been shown to be localized to the division plane in B. subtilis and C. crescentus. Although FtsZ has no apparent homology to known eukaryotic cytoskeleton proteins, it appears to function as a cytoskeletal element in bacteria. It is not known how FtsZGTP recognizes a specific site on the cell to initiate its localized polymerization, although the Min proteins have been implicated in site selection (de Boer et al., 1989). There are at least two systems that monitor the “start division” decision: the Min system prevents division at sites other than the equator (Cook et al., 1987; de Boer et al., 1989, 1990) and the SOS system detects disruptions in DNA replication and, through the SulA protein, prevents division in a cell unable to complete the duplication of its chromosome (Huisman et al., 1984; Freud1 et al., 1987). In both of these cases, the FtsZ protein is the target of the cell division inhibitors (de Boer et al., 1989; Bi and

Review: Protein Localization

in the Bacterial Cell

043

Figure 2. A Model of the Cell Division Apparatus Site of a Dividing E. coli Cell

at the lnvagination

The FtsZ ring interacts with the integral membrane proteins, FtsA and Ftsl, as described in the text. Two other division proteins, FtsL and FtsQ, are anchored in the inner membrane, but the major portion of the proteins is in the periplasm.

Lutkenhaus, 1990a, 1990b), suggesting that the formation of a FtsZ annular ring is a key event in coordinating DNA replication and cell division. Once FtsZ-GTP forms an annular ring around the circumference of the cell at the cytoplasmic face of the inner membrane, it recruits a complex of several proteins to the septal site. Thus far, two integral membrane proteins, Ftsl (Pbp3) and FtsA, have been implicated in cell division (Pla et al., 1990). The requirement for both of these proteins occurs after FtsZ polymerization initiates the division process at the septal site. The Ftsl protein catalyzes the synthesis of the septal peptidoglycan wall (Ishino and Matsuhashi, 1981). Although the biochemical function of FtsA is not known, it is required throughout the septation process, and it apparently interacts with the Ftsl protein (Tormo et al., 1986). There is indirect evidence that FtsA (Descoteaux and Drapeau, 1987; Dai and Lutkenhaus, 1992; Dewar et al., 1992) and Ftsl (Ayalaet al., 1988) interact with FtsZ. Twoadditional membrane proteins, FtsL(Guzman et al., 1992) and FtsQ (Carson et al., 1991) are required for septum formation. These proteins are present in very few copies (20-40) per cell, and, although anchored in the membrane, they reside predominantly in the periplasm. Analysis of fi.sL mutants suggests that FtsL might couple cell division and peptidoglycan synthesis. Based on the locations and interactions of these proteins, a mode! of the cell division machine, albeit incomplete, is shown in Figure 2.

and BUD5) direct bud site selection (Bender and Pringle, 1989; Chant and Pringle, 1991; Chant and Herskowitz, 1991; Chant et al., 1991). The expression of BUD7, BUDP, and BUD5 in either a or a haploids or in ala, ala, or ala diploid mother cells is required for bud site selection. The additional expression of BUD3 and BUD4 in a or a haploid cells or in a/a and a/a diploids results in the axial budding pattern. The repression of BUD3 and BUD4 expression in a/a diploids causes bipolar bud site selection. In cells lacking BUD7, BUDP, or BUDS, the complete default pattern is random bud site placement. The events subsequent to bud site selection that are required to build the cell division apparatus are not dependent on the BUD gene hierarchy. Mutants in which bud site selection is random grow well and thrive. When a/a diploid strains are grown under conditions of nutrient deprivation, the cells must forage for food, and an additional positional constraint is imposed on the division process: polar division is directional so that a chain of cells grows in one direction yielding pseudohyphae (Gimeno et al., 1992). In fact, the rigid constraints on division site selection in yeast can only be easily rationalized in the case of directional polarity to yield pseudohyphal formation. The function of the BUD gene products is to guide the proteins essential for bud emergence to a specific site on the cell surface (Chant and Pringle, 1991; Chant and Herskowitz, 1991). Several of the BUD genes have been sequenced and their homologs identified. The BUD7 gene is a homolog of the small Ras-like GTP-binding proteins, such as Rho and Rat (Hall, 1992) and the Rab family (Pfeffer, 1992), which are involved in vesicle targeting and morphogenesis. BUD2 is a homolog of GTPase-activating proteins, and it is believed to stimulate the intrinsic GTPase of BUD7 (H.-O. Park, J. Chant, and I. Herskowitz, personal communication). BUD5 is a homolog of GDPGTP exchange proteins (Chant et al., 1991; Bender and Pringle, 1989). It has been proposed that the previous bud site leaves a membrane marker adjacent to the bud scar that is used by the BUD genes for the site of new bud formation. In haploid cells, membrane association of BUD3 and BUD4 at this site would establish the axial pattern of bud site selection (Chant and Herskowitz, 1991). It has, in fact, recently been demonstrated that the BUD3 protein is localized to the neck of the bud (J. Chant and J. Pringle, personal communication). The biochemical reactions and

random

The Yeast Cell Bud site selection in the yeast S. cerevisiae, as in the selection of the division plane in E. coli, requires the localization of a protein complex to a specific site at the surface of the mother cell. Under normal growth conditions, the selected bud site is determined by the mating type locus MAT. In haploids, bud site selection is axial, occurring adjacent to the previous site of cell division. In diploid cells, bud site selection is polar, occurring either adjacent to the previous site of cell division or at the opposite pole of the cell (Figure 3). Five genes (BUD7, BUDP, BUD3, BUD4,

A

bipolar BUD1 BUD2 BUD5

-

axial BUD3 BUD4

Figure 3. The Pattern of Bud Site Selection in S. cerevisiae The genes involved in either bipolar or axial bud site selection (Chant and Herskowitz, 1991) are indicated and are described in the text.

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and/or CDC42

guanine nucleotide exclmlge factor HUD SITE

Figure 4. The Pathway of Bud Site Selection in S. cerevisiae The model considers the suggested biochemical functions of the BUD gene products in axial bud site selection, as described in the text.

protein interactions that are believed to mediate axial bud site selection are diagrammed in Figure 4. The active GTPbound form of BUD1 appears to interact with CDC24 (Bender and Pringle, 1989) and this complex is guided to the BUD3 and BUD4 axial site in the mother cell membrane. CDC24 is a Ca2+-binding protein that is needed to restrict growth to a specific site on the cell. Since BUD2 may be a GTPase-activating protein for BUDl, it might either be located at the bud site or travel along with BUD1 In Figure 4, BUD2 is shown located at the bud site. Because budl- and b&2- mutants have the same phenotype (random bud site selection), it may be that if BUDl-GTP hydrolysis to BUDl-GDPcannot be activated bytheBUD2 GTPase-activating protein activity, then BUDl-GTP remains stuck at the bud site. Its recycling could be necessary either to choose the proper bud site or to commit the cell to using this particular site. Once the bud site is selected, the polarized movement of secretory vesicles is facilitated by the establishment of actin cables directed toward that site (Novick and Botstein, 1985; Drubin et al., 1988). How might the BUD gene products co-opt cytoskeleton components for polar organization at the bud site? A structural protein, BEMl, which has actin-organizing domains, is essential for bud formation (Chenevert et al., 1992). BUD1 and a second small GTP-binding protein, CDC42, which is involved in bud formation, are believed to interact with BEMl, supporting the argument that the BUD proteins communicate with cytoskeletal elements (Chant et

al., 1991). beml mutants exhibit disrupted actin organization (Chenevert et al., 1992). BEMl and a second actinassociated protein that functions in bud formation, ABPl (Drubin et al., 1990), could interact with the bud site and with the actin cytoskeleton to direct polarized trafficking of vesicles to the emerging bud. Division site selection in bacteria and bud site selection in S. cerevisiae both use GTP-binding proteins. Some of the actions of these proteins are held in common, such as their role in recognizing markers (albeit poorly understood in both cases) at the cell surface and the controlled transition between active and inactive formsof the protein. However, their roles are fundamentally different: in the case of E. coli, the FtsZ-GTP protein not only sequesters to the site of division but might be a cytoskeletal monomer that polymerizes to form the contractile ring. The activated GTP-bound form of the bacterial protein functions in a manner analogous to GTP protein monomers in tubulin assembly. The BUDl-GTP protein of yeast, on the other hand, although sequestered to the bud site, functions in vesicle targeting to the site of daughter cell growth and participates in a cascade of events that activates and orients the cytoskeletal structure for the complex morphogenesis that accompanies bud emergence and division. The Chemosensory

Apparatus

The Bacterial Cell Because the actual mechanics of cell division occur at a defined zone, generally in the middle of a rod-like cell, and at axial or polar sites in budding yeast, it is not surprising that the proteins involved in the cell division machinery are transiently localized to these specific cell sites. However, the observation that other kinds of biochemical complexes are also localized to specific sites on the cell was unexpected (Maddock and Shapiro, 1993; Alley et al., 1992, 1993). The bacterial chemoreceptor and accompanying proteins that are necessary for chemosensory transduction were found in both E. coli and C. crescentus to be confined to the poles of the cell (Maddock and Shapiro, 1993; Alley et al., 1992, 1993). Chemoreceptors, which are highly conserved among all bacteria in which they have been studied, are integral membrane proteins with two transmembrane domains (Figure 5; see Parkinson, 1993 [this issue of Cc/d). The periplasmic domain of the chemoreceptor interacts with ligand. The occupancy of the ligand domain with either an attractant or a repellent is thought to change the conformation of the chemoreceptor that mediates the transmission of a chemotactic signal to the flagella rotor. The signal to the flagella facilitates net movement of the bacterium toward or away from high concentrations of the ligand. This signal transduction pathway is a well-characterized phosphorelay involving several cytoplasmic proteins (Parkinson, 1993). Two of these proteins, a histidine phosphokinase (CheA) and a protein of unknown biochemical function (Chew), were shown in vitro to form a long-lived ternary complex with the chemoreceptor (Gegner et al., 1992). The complex of the bacterial chemoreceptor and

Review: Protein Localization 845

in the Bacterial Cell

A

Figure 5. Localization

of Chemoreceptors

to the Pole of the E. coli Cell

(A) lmmunoelectron micrographs of longitudinal sections of E. coli after incubation with anti-Tsr antibodies (from J. R. Maddock). Goat anti-rabbit 10 nm colloidal gold conjugates reveal the position of the Tsr chemoreceptors on the ceil sections. (6) A schematic of polar clustering of the chemoreceptors (methyl-accepting chemotaxis protein [MCP]) in complex with CheA and Chew. In the absence of any one of the three members of the complex, polar localization fails to occur (Maddock and Shapiro, 1993).

Cell 646

the CheA kinase is analogous to many eukaryotic receptor kinases, which combine both functions in one protein. Analysis of allele-specific second site suppressors identified a 45 amino acid highly conserved domain as the site in the cytoplasmic portion of the chemoreceptor that interacts with CheA and Chew (Liu and Parkinson, 1991). Deletions that remove the highly conserved domain cause the truncated chemoreceptor to lose polar localization. Using antibodies to the E. coli chemoreceptors and to CheA and Chew, it was observed that these proteins form clusters at the inner membrane and that these clusters are primarily at the cell pole (Figure 5A; Maddock and Shapiro, 1993). In strains deleted for all chemoreceptors, CheA and Chew are distributed randomly in the cytoplasm, and in strains deleted for CheA and Chew, the chemoreceptors are distributed randomly in the cytoplasmic membrane. Further analysis of mutant strains revealed that a binary complex between Chew and the chemoreceptor localize to the cell pole in the absence of CheA, but that in strains that lack Chew, the chemoreceptor and CheA form a membrane-associated complex, but it is not localized to the pole (Maddock and Shapiro, 1993). Two additional cytoplasmic proteins, CheY and CheZ, participate in the transfer of the signal to the flagella rotors. Neither CheY nor CheZ is required for polar localization of the chemoreceptors. The observed polar distribution of the bacterial chemoreceptor complexes raises two questions: what is the functional significance of localizing these receptor complexes to the cell pole, and what is the mechanism of polar localization? Considering these questions raises our appreciation of the complexities of the bacterial cell to a new level. Why should a very small cell without an apparent need for intracellular trafficking nevertheless separate complexes of distinct biochemical function to sequestered regions of the cell? It may be that sequestering provides a more advantageous biochemical milieu and that the pole of the cell is simply a convenient site to deposit this particular signal transduction machine. The concept of localized clusters of functionally interdependent proteins is further supported in this case by the colocalization of an additional component of the chemosensory apparatus. Several chemoreceptors associate with periplasmic binding proteins that first interact with the relevant ligand. One of these periplasmic binding proteins, MalE, which functions as a bridge between ligand and the Tar chemoreceptor, is also located at the cell pole (Boos and Staehelin, 1981; Maddock and Shapiro, 1993). Given the range of the internal signal (Segall et al., 1985) it is apparently not essential for the receptors to be spatially separated at the cell poles of normal cells for a temporal sensing mechanism to be operative. However, in longer cells, the sensing of a spatial gradient may be relevant. How does a cell without a known cytoskeleton sequester cellular components in specific regions of the cell? There are at least two routes by which the ternary complex could become localized at the cell pole; both rely on some mechanism for polar retention. In the first route, the newly synthesized chemoreceptor is inserted directly into a site at the cell pole via a polar secretory signal. Lateral diffusion

away from that site in the membrane might be prevented by complex formation with the Chew and CheA proteins. Alternatively, the new chemoreceptors are inserted randomly in thecytoplasmic membrane, and membrane diffusion would allow ternary complexes of chemoreceptor, CheA, and Chew to travel to, and then be retained at, the cell pole. These alternatives suggest that polar components as yet unidentified entrap the ternary chemoreceptor complex. Because the bacterial cell poles originate as the site of cell separation, a polar marker could be laid down at that time, contributing to the notion of a continuous line of asymmetric surface markers. This is directly analogous to a membrane marker deposited adjacent to the yeast bud scar, which dictates the site of the next bud emergence. Other examples of proteins that are targeted to the poles of bacterial cells include a lectin, hemagglutinin, in aggregating Myxococcus xanthus (Nelson et al., 1981) and the surface protein IcsA in the pathogen Shigella flexneri (Goldberg et al., 1993). The myxobacteria undergo a striking morphogenesis if starved for nutrients (Kaiser and Losick, 1993). The cells swarm to an aggregation center and form a multicellular fruiting body. Hemagglutinin, which is detected in aggregating cells but not in vegetatively growing cells, is localized in surface patches, predominantly at the poles of the cells (Nelson et al., 1981). Although the function of hemagglutinin is not known, it has been suggested that its polar position may mediate the end-to-end interactions of aggregating cells. In the case of the invasive pathogen S. flexneri, the bacterium enters the host cell by phagocytosis and then induces the assembly of actin filaments at one end of the bacterial cell. This actin tail propels the bacterium within and across cell boundaries. The polymerization and attachment of the actin tail to one end of the bacterial cell are mediated by a membrane protein, IcsA, that is located at the pole of the free-living, and the intrahost, bacterial cell. Other invasive pathogens, such as Listeria monocytogenes, also use the actin cytoskeleton of the host to move through the cell and to spread to adjacent cells (Dabiri et al., 1990). L. monocytogenes interacts with actin filaments by virtue of a protein, ActA, that is tethered to the cytoplasmic membrane but is predominantly extracellular (Kocks et al., 1992). The ActA protein in the free-living L. monocytogenes and in the host tissue is found at one pole and on the sides of the cell but is excluded from the opposite pole (C. Kocks and P. Cossart, unpublished data). Neither the IcsA nor the ActA protein can be detected at the septating site, suggesting that the new pole lacks these proteins. These examples of localized bacterial proteins suggest that the site of protein aggregation at a specific site at the cell surface mediates interaction of the cell with extracellular events. These events include ligand detection, oriented interaction with other cells, and hitchhiking rides on the cytoskeleton of foreign cells. The Yeast Cell The deposition of receptors to the cell pole also occurs during mating of S. cerevisiae a and a cells. Haploid a

Review: Protein Localization 047

in the Bacterial Cell

Figure 6. B. subtilis Cell Division The figure depicts both vegetative cell division and the asymmetric cell division that initiates sporogenesis. The Ftd protein is an essential component of both divisions (Beall and Lutkenhaus, 1991).

asymmetric division

symmetric division

cells localize the a receptor to the position on the cell nearest the highest concentration of the pheromone produced by a cells (Jackson et al., 1991). When an a cell is activated by the binding of pheromone to the membranebound a receptor, a signal transduction pathway is initiated that results in a cascade of gene expression. The localization of the a receptor to one site on the cell surface in response to pheromone is accompanied by a change in cell shape (creating a shmoo with an elongated cell pole) and by a reorganization of the cytoskeleton to provide a new cellular axis of symmetry. The BEMl protein that is used for the organization of the actin cytoskeleton in bud site selection is also required for the cell polarization in response to mating pheromones (Chenevert et al., 1992). In this case, the morphogenetic marker at the cell pole recognized by BEMl-associated proteins might be the a receptor itself (Chenevert et al., 1992). The localization of the a receptor only occurs upon exposure to pheromone. The receptor is distributed randomly around the cell prior to receptor-ligand interaction (Jackson et al., 1991). In the bacterial cell, it has not been possible to detect uniform distribution of the chemoreceptors, suggesting either that they are localized to the cell pole upon synthesis or that they rapidly migrate to the pole, where they are retained upon interaction with other components of the signal transduction pathway. At low concentrations of pheromone, the gene encoding the a receptor is induced, as is a gene required for fusion, FUS1. However, the protein products of these genes are only localized to a specific site if there are concentrations of pheromone high enough to cause polar projection formation. It is not known whether it is the newly synthesized a receptor that is localized or whether the existing receptors are rearranged to form a cap at the pole of the cell. The a receptor and the FUSl protein share the pole of activated a cells with another protein, SPA2 (Snyder, 1989). This protein is also found at the pole of the emerging bud during vegetative growth (Gehrung and Snyder, 1990). Analysis of spa2 mutant cells suggests that this protein plays an important role in cellular morphogenesis during the mating process, yet spa2 mutants are still able to localize the a receptor upon exposure to pheromone. This result suggests that a complex between the a receptor and SPA2 is not necessary for receptor localization,

in contrast with that seen with the E. coli chemoreceptor and the CheA and Chew proteins. Mammalian Neutrophils Polymorphonuclear neutrophils contain transmembrane receptors for many different ligands on their cell surface. Two of these receptors, those recognizing the Fc domain of immunoglobulin G (Walter et al., 1980) and those for the plant lectin concanavalin A (Weinbaum et al., 1980), are localized to the tip of the cellular projection or pseudopodia formed in response to the chemoattractant. Although the mechanism of localization is not understood, in chemoattractant-induced pseudopods these clustered receptors activate G proteins and initiate a signal transduction cascade that results in directed cell motility. It has been proposed that the clusters of chemoreceptors at the pole of the pseudopod extension stabilize small actin oligomers that then form actin filaments oriented along the axis of the pseudopods (Schwartz and Luna, 1988). Asymmetry

and Cellular Differentiation

Formation of an Asymmetrically Positioned Septum Initiates Cell Differentiation in Bacillus Sporogenesis Cell division in 6. subtilis vegetative cells is equatorial, and, as is the case in E. coli (Bi and Lutkenhaus, 1991), the FtsZ protein initiates the septation process by forming a contractile ring at the incipient division site (Beall and Lutkenhaus, 1991; X. Wang and J. Lutkenhaus, unpublished data). In addition to FtsZ, another cell division protein initially identified in E. coli, FtsA, whose function is required subsequent to the establishment of the contractile ring, is also required for B. subtilis vegetative division (Beall and Lutkenhaus, 1992). When B. subtilis cultures are starved for nutrients, heat-resistant, refractile spores are formed (Kaiser and Losick, 1993). A key event in this process is asymmetric septum formation that yields progeny, a forespore and a mother cell, which differ structurally and functionally(Figure 6). The morphogenesis of the forespore into a spore requires transcripts provided by the mother cell chromosome that encode proteins that are used to build the spore coat (Figure 7). Transcripts provided by the forespore encode proteins that bind to the

Cell 848

spoIIGA

, spoIIGB

predivisiond

cell

spoIlAA

43 AA

forespore chromosome and help to build the inner structure of the spore. Once the spore is completed, the mother cell self-destructs (Figure 6). To effect these distinct programs, the cell committed to the sporulation pathway produces two new o factors, qF and pro-oE, prior to septum formation, which will ultimately direct the transcription of different genes in the forespore and mother cell (Margolis et al., 1991; reviewed in Losick and Stragier, 1992). A central question is how the uniform distribution of d factors is spatially redirected so that the forespore and mother cell formed by the deposition of the asymmetric septum have different o factor activities. The answer to this question provides an elegant example of the codependence of morphogenesis and transcriptional regulation. Beall and Lutkenhaus (1991,1992) have shown that the formation of the asymmetric septum in the sporangium requires the same cell division proteins, FtsZ and FtsA, as are required for equatorial vegetative cell division. The factors that direct the contractile FtsZ ring to a new position in the sporulating cell are unknown, but the localization of this ring represents the first visible expression of asymmetry in this specialized cell differentiation pathway (see Figure 6). If the asymmetric septum fails to form, the two new o factors, uF and pro&, remain inactive, and the transcription of all subsequent sporulation-specific genes fails to occur (Beall and Lutkenhaus, 1991; Losick and Stragier, 1992). Thus, the assembly of the septum is intimately related to the differential activation of the o factors that initiate the morphogenetic pathways in the mother cell and the forespore. The diagram shown in Figure 7 portrays a model that takes into account the known interactions among proteins whose genes are expressed in the early sporangium during the formation of the asymmetric septum. The gene

, spolIAB

spolIAC

Figure 7. The Role of Septum Formation in B subtilis Sporogenesis The figure shows the role of septum formation in the differential activation of oF in the forespore and oE in the mother cell during early B. subtilis sporogenesis. Inactive of and pro-6 are produced in the predivisional cell. All furthereventsaredependenton FtsZfunctionand the establishment of the asymmetric septum, as described in the text. Active proteins are indicated in outlined letters.

encoding oF, spollAC, is the final gene in an operon that encodes twoother proteins, AA and AS Analysisof strains containing mutant alleles of each of these genes suggests that the A6 gene product functions as an anti-o factor and inhibits qF activity. When the AA protein is activated following septation, it appears to function as an inhibitor of AB (Losick and Stragier, 1992). The AA protein cannot inhibit AB prior to septation, and thus oF remains in an inactive state in the predivisional cell. Once septation occurs, the AA protein is activated, but only in the forespore compartment. Active AA thus inhibits AB function, and forespore dF is activated. What feature of the septum or consequence of septum deposition confers directional activation of aF? Two elements may play a role in directional activation: the insertion of proteins on one or the other face of the septal membrane and the marked difference in the volume of the two compartments. Because a mem-

Caulobactrr

Figure 8. A Comparison of Cell Division in C. crescentus Asymmetric Sporulation Division in B. subtilis

and the

Review: Protein Localization 849

in the Bacterial Cell

different chromosomes are interdependent yield differentiated cell types.

[q differential

tranXription

ga 8

e 5‘

o Stalked cell

& Predivisional cell

Figure 9. Events in the Expression Caulobacter Predivisional Cell

differential protein targeting

[ej

of Polarity and Asymmetry

in the

brane protein, SpollE (Gholamhoseinian and Piggot, 1989) has been shown to be required for normal septum formation and for cF activation, it has been suggested that the directional deposition of SpollE in the septal membrane facing the forespore provides the initiating signal for oF activation in the forespore compartment (Margolis et al., 1991; llling and Errington, 1991). Meanwhile, a different series of events is happening on the mother cell side of the newly formed septum. While uF remains in an inactive state in the mother cell, crE is activated (see Figure 7). When cE is produced in the predivisional cell, it exists as an inactive proprotein with an extra 27 amino acids at its N-terminus (LaBelI et al., 1987). Proteolytic cleavage of the N-terminal 27 amino acids by the protease encoded by the adjacent spollGA gene contributes to the activation of cE. Active cE is detected only in the mother cell compartment (Driks and Losick, 1991), suggesting that eE activation occurs only after the formation of the septum. It has, in fact, been argued that the proteolytic conversion of pro-GE to active crE is specific to the mother cell compartment (Errington et al., 1990). In B. subtilis cells carrying a mutant f&Z allele and thus unable to assemble a septum, the pro-oE is not processed to its active form (Beall and Lutkenhaus, 1991). Assuming that pro-GE is processed only in the mother cell compartment, we are again faced with the problem of a cell typespecific biochemical event. A cF transcriptional event on the forespore side of the septal membrane is required to activate oE in the mother cell. It has been proposed that an unknown factor is produced under the direction of dF in the forespore compartment, and this factor interacts with the septal membrane to produce a directional signal that stimulates the membrane-bound SpollGA protease to cleave pro& in the mother cell compartment (Losick and Stragier, 1992). The genes transcribed by oE from the mother cell chromosome encode structural components of the spore that are then added to the septal membrane from the mother cell side of the sporangium. In the final stages of sporogenesis, two new o factors, cG and cK, are activated in the encapuslated sporangium and in the mother cell, respectively (see Kaiser and Losick, 1993). In this manner, the evolving structure of the sporangium and the transcription of specific sets of genes from two

events that

Asymmetric Distribution of Proteins in the Caulobacter Predivisonal Cell Yields Different Progeny Cells Both Bacillus and Caulobacter are capable of a cell division that yields different progeny. The Caulobacter progeny swarmer and stalked cells are structurally distinct, and the two cells differ in their pattern of transcriptional activity (Newton and Ohta, 1990; Gober and Shapiro, 1991). Only the swarmer cell, with a single polar flagellum, is motile and responds to chemotactic stimuli. An additional fundamental difference between these progeny cells is that only the chromosome in the stalked cell can initiate DNA replication (Degnan and Newton, 1972; Marczynski et al., 1990; Marczynski and Shapiro, 1992). The differentiation of progeny cells is an inherent part of the Caulobacter cell cycle but is induced by environmental changes in Bacillus. The fundamentally different mechanisms used by Bacillus and Caulobacter to affect the differentiation of progeny cells is diagrammed in Figure 8 and described in more detail for Caulobacter in Figure 9. In the case of Bacillus, differential transcription of the newly replicated chromosomes and the consequent morphogenesis follow the deposition of a septum near one pole of the cell. The release of the spore is accompanied by the degradation of the mother cell. In contrast with the pattern of cell differentiation during sporogenesis in Bacillus, many of the distinct characteristics of swarmer and stalked cell progeny are established prior to cell division, by virtue of the differential distribution of gene products to the two poles of the predivisional cell (Gober et al., 1991b; Alley et al., 1992, 1993). The Caulobacter progeny stalked cell has a strikingly dif-

Asymmetry

Figure 10. The Caulobacter

Cell Cycle

The biochemical events that lead to the asymmetric expression and localization of proteins are shown, The 9 structures indicate replicating DNA.

Cell 850

ferent fate than the Bacillus mother ceil. The stalked cell is not degraded, but functions as a stem cell, giving rise to a swarmer cell and regenerating a stalked cell at the next division cycle. The predivisional cell has polar domains that differ from one another; one pole has a stalk and the other pole has a newly assembled flagellum, pili, and chemotaxis machinery (Figure 9). Several groups of proteins are differentiallysegregated to the progeny swarmer and stalked cells. Segregation of proteins to these cells is a key event that gives the two progeny different identities. This asymmetric distribution of proteins appears to be due to at least two mechanisms: differential transcription of the newly replicated chromosomes in the predivisional cell (Gober et al., 1991a) and localization of specific proteins to only one pole of the predivisional cell and their consequent appearance in the progeny cell arising from that pole (Alley et al., 1992, 1993). Because the Caulobacter cell cycle includes the morphogenesis of one cell type into another, the proteins at the pole of the swarmer cell are degraded when it differentiates into a stalked cell (Figure 10). Differential Transcription Several genes encoding flagellar structural proteins are transcribed in the predivisional cell. The products of these genes appear only in the progeny swarmer cell. Transcriptional fusions to reporter genes were used to determine whether these flagellar genes are preferentially transcribed from the chromosome in the swarmer pole of the predivisional cell. Protein products from these fusions, labeled in the predivisional cell, were detected by immunoassay in the progeny swarmer cell (Gober et al., 1991a). Segregation to the swarmer cell of reporter proteins expressed from the flagellar transcriptional fusions does not depend on sequences within the flagellar messenger RNA (mRNA), but on the upstream regulatory regions (Gober et al., 1991a; Gober and Shapiro, 1992). Reporter genes under the control of their own promoters yield protein products that segregate to both progeny cells (Gober et al., 1991a). The presence of the reporter gene protein in the swarmer cell, but not in the stalked cell progeny, is a biochemical “snapshot” of transcription in the predivisional cell (when labeling occurred), suggesting that the chimeric gene is specifically transcribed from the chromosome residing in the swarmer portion of the predivisional cell. Electron micrographs of the predivisional cell have revealed a region at the incipient division plane that excludes colloidal gold markers (J. R. Maddock, unpublished data). These observations suggest that a nonmembranous barrier may form between the two halves of the predivisional cell late in the cell cycle. This barrier could explain why the reporter as well as natural gene products appear not to diffuse throughout the predivisional cell. The genes that appear to be transcribed from the chromosome residing in the swarmer portion of the predivisional cell are all transcribed late in the cell cycle. They also share similar cis-acting regulatory elements, including a os4 promoter and essential enhancer and integration host factor-binding sites that reside either 5’ or 3’ to the

promoter (Gober et al., 1991 a; Gober and Shapiro, 1992). Given the shared regulatory elements among these differentially transcribed genes, it is possible that the factors that bind to these elements or proteins that activate these factors might be consigned to the swarmer portion of the predivisional cell. As described below, membrane proteins and cytoplasmic proteins that bind to them have been shown to be localized to the incipient swarmer pole of the early predivisional cell, prior to the formation of any barrier between the two halves of the cell (Alley et al., 1992,1993). Another asymmetric characteristic of the predivisional cell that may contribute to the differential transcription of the chromosomes is that the physical properties of the two chromosomes appear to differ. The chromosomes markedly differ in their sedimentation coefficient (Evinger and Agabian, 1979; Gober and Shapiro, 1991; Swoboda et al., 1982). The more highly condensed chromosome is inherited by the swarmer cell. When the swarmer cell differentiates into the stalked cell later in the cell cycle, the chromosome sedimentation coefficient changes to that seen in the stalked cell following cell division (Figure 10). These structural differences between the two chromosomes may allow differential access to transcription factors As shown diagrammatically in Figure 8, the chromosomes that segregate to the B. subtilis forespore and mother cell also differ in their condensation state (Setlow et al., 1991). Marked condensation of the forespore nucleoid occurs several hours prior to the expression of the sporespecific genes but fails to occur in spo0 mutants. There is no evidence, however, that the difference in chromosome condensation in the sporangia contributes to differential transcription. DNA packaging into the small spore volume might be the sole requirement of the condensed chromosome. It is not apparent, however, why the chromosome in the Caulobacter swarmer cell should house a condensed nucleoid. Any of the proposed mechanisms for the differential transcription of the chromosomes in the Caulobacter predivisional cell rely on a preexisting asymmetry. Thus, differential transcription in the predivisional cell is a consequence, not a cause, of cellular asymmetry and the resulting different progeny cells. Protein Localization The single Caulobacter flagellum appears de novo at the pole opposite the stalk (see Figure 9). The initiation of flagellar biogenesis at the pole of the predivisional cell requires protein localization. The components of the flagellum are added to the nascent structure in a cell proximal to cell distal order, beginning with the transmembrane rotor, continuing with the hook, and ending with the distal filament. It is not known whether the polar site for flagellum assembly isselected bythe insertion of asingle membrane protein, such as the M ring of the rotor, and then followed by the localized assembly of the rest of the flagellar components in a passive cascade from that initial event or whether several flagellar proteins are independently targeted to the cell pole.

;ge;ew:

Protein Localization

in the Bacterial Cell

Figure 11. Localization of Chemoreceptors at Different Stages of the C. crescentus Cell Cycle

A. Wild-Type Chemoreceptor

(A) Wild-type chemoreceptors. (6) Chemoreceptors expressed from a multicopy plasmid. (C) Mutant chemoreceptors lacking a proteolyticsignal attheC-terminus (Alleyet al., 1993). The black dots in all three panels represent the cellular location of the chemoreceptors.

B. Overexpressed Chemoreceptor

0

0

i I 0 c M/(1FQ c ( 1

C. Chemoreceptor with a C-terminal Deletion

t M C @ SYNTHESlS 1

Motile swarmer cells are chemotactically competent, and it has been shown that proteins involved in chemotaxis are synthesized before cell division and then segregated to the progeny swarmer cell (Gomes and Shapiro, 1984; Alley et al., 1992). This asymmetric segregation to one progeny cell is due to the fact that upon de nova synthesis the chemoreceptors are targeted to the incipient swarmer

pole of the predivisional cell (Figure 11A; Nathan et al., 1988; Alley et al., 1993). The highly conserved domain near the C-terminus of the chemoreceptor is essential for polar localization (Alley et al., 1993). Based on the observation that E. coli chemoreceptors are targeted to the cell pole as a complex containing the CheA and Chew signalrelay proteins bound at the highly conserved domain (Mad-

Cell 852

dock and Shapiro, 1993) it is likely that a similar mechanism is operative in the case of Caulobacter. Observation of immunogold-tagged chemoreceptors during the cell cycle (Figure 11A) revealed that wild-type chemoreceptors are present at the flagellated pole of the predivisional cell, are lost from that pole when swarmer cells differentiate into stalked cells, reappear at the incipient swarmer pole of the predivisional cell, and are detected at the pole of the progeny swarmer cell, but absent in the progeny stalked cell (Alley et al., 1993). When an E. coli chemoreceptor gene is expressed in Caulobacter, the E. coli chemoreceptors are targeted to the pole of the swarmer ceil, but they are not turned over during the cell cycle (Alley et al., 1992, 1993; J. R. Maddock and M. R. K. Alley, unpublished data). This result suggests that theCaulobacterCheAand Chew proteinscan interact with the E. coli chemoreceptor. Of greater significance, these results also suggest that an inherent signal in the Caulobacter chemoreceptor causes its asymmetric appearance at the incipient swarmer pole. Comparison of the amino acid sequences of the E. coli and the Caulobacter chemoreceptors reveals that a short sequence present at the C-terminus of the Caulobacter chemoreceptor is absent from the E. coli protein. Deletion of this 14 amino acid sequence at the C-terminus of the Caulobacter chemoreceptor yields cells with bipolar distribution of the chemoreceptor (Figure 1lC). Mutant chemoreceptors that have the 14 amino acid deletion at the C-terminus are not turned over during the swarmer-to-stalked cell transition and thus appear at both poles of the predivisional cell and in both of the progeny cells. If the wild-type chemoreceptor is moderately overexpressed (Figure 11 B), it transiently appears at the stalked pole of the predivisional cell, but localized proteolysis results in a progeny stalked cell that lacks chemoreceptors. Thus, a temporally and spatially regulated proteolytic event contributes to the asymmetry observed during the wild-type Caulobacter cell cycle. The proteolysis observed during the swarmer-to-stalked cell transition extends to other chemotaxis proteins, including the methyltransferase and the methylesterase (Gomes and Shapiro, 1984). The release of the flagellum, loss of polar pili, and change in the sedimentation coefficient of the nucleoid coincide with the turnover of the chemotaxis proteins during the cell type transition and may depend, in part, upon specific proteolytic events. It is formally possible that the newly synthesized chemoreceptors are initially randomly distributed around the cell, but only those at the poles are immune to proteolysis. However, it is clear that proteolysis of laterally distributed chemoreceptors is not the mechanism of polar localization since in the absence of proteolytic signals in the E. coli chemoreceptor or in the mutant Caulobacter chemoreceptor, these proteins still are targeted to the cell pole. It is only asymmetry that is lost. The mechanisms used to target and retain the chemoreceptor clusters to bacterial cell poles are not known. Because the cell poles are the site of an earlier cell division, it may be that a recognition patch is left, as a sort of bud scar, at the cell pole. Thus, cells may retain a positional

memory, as in the case of BUD3 and BUD4 localization to the site of previous bud formation in yeast. Selective retention of proteins is used by mammalian cells for polar localization, although the direct recruitment of vesicles to a given site on the cell surface is a more ubiquitous mechanism for generating localized plasma membrane domains in polarized epithelial cells (Hopkins, 1991; Nelson, 1992). An example of selective retention is the deposition of Na+/K+-ATPase at the plasma membrane of polarized MDCK cells (Hammerton et al., 1991). Newly synthesized enzyme is delivered to both the basal-lateral and the apical domains, but the apical Na+/K’-ATPase is inactive and is rapidly removed from that cell surface, analogous to the rapid removal of the overexpressed chemoreceptor at the stalked pole of the Caulobacter predivisional cell. Retention of Na+/K+-ATPase in the basal-lateral membrane involves linkage to the underlying membraneassociated cytoskeleton, which is also localized to this membrane domain. Direct recruitment relies on an intrinsic targeting signal in the amino acid sequence within the cytoplasmic or extracellular domain of the protein. Targeting and retention signals do not appear to be mutually exclusive. A membrane anchor, glycosylphosphatidylinositol, is attached to proteins that are targeted to the apical pole of MDCK cells (Lisanti and Rodriguez-Boulan, 1990). There is evidence that this hydrophobic anchor attaches to glycosphingolipids that are preferentially clustered in the trans-Golgi network and incorporated into transport vesicles that are delivered to the apical membrane of MDCK cells (Simons and Wandinger-Ness, 1990). Transfer of the glycosylphosphatidylinositol anchor to a protein that is not normally found in the apical membrane of MDCK cells in fact confers the ability to target that protein to the apical domain (Brown et al., 1989; Lisanti et al., 1989, 1990). Thus, in this case, it is not the primary sequence of the protein but rather an additional moiety to the protein that confers targeting specificity. A cytoplasmic domain with a structural motif found in several targeted proteins has been shown to mediate targeting of these proteins to the basal-lateral membrane of polarized epithelial cells (Mostov et al., 1986; Casanova et al., 1991; Collawn et al., 1991). Sorting of this class of proteins may involve a process of clustering in the transGolgi network similar to the formation of coated vesicles during endocytosis (Pearse and Robinson, 1990). As in the case of bud site selection and establishment in yeast, directional movement of vesicles from the Golgi complex to different cell surface domains appears to involve cytoskeletal proteins and GTP-binding proteins (Nelson, 1992). Actin filaments mediate bud formation in yeast, and both actin filaments and microtubules may facilitate traffic between the trans-Golgi network and the cell surface in polarized epithelial cells. In bacterial cells, the cytoplasmic membrane itself appears to be the conduit for the membrane protein, in complex with other cellular proteins, that segregates to the cell pole. In all these cases, ultimately, the protein or protein complex must dock with and/or be retained at some sort of recognition site. In mammalian epithelial cells, cell-cell and cell-substratum interactions

Review: Protein Localization 053

in the Bacterial Cell

mediate the establishment of localized recognition sites that set up different membrane domains (Nelson, 1992) whereas in mating yeast cells, the localized concentration of a pheromone contributes to the polar localization of the a receptor and the axis of symmetry of the cytoskeleton components. Although extracellular signals appear to mediate polar localization of proteins in mammalian epithelial cells and in mating yeast cells, bud site selection in yeast, asymmetric septum formation during Bacillus sporangenesis, and the polar deposition of chemoreceptors in Caulobacter and E. coli appear to be due to cell-intrinsic functions. Acknowledgments I thank I. Herskowitz, R. Losick, and J. Lutkenhaus for helpful discussions and J. Nelson for comments on the manuscript. A portion of the work discussed in this review was supported by United States Public Health Service grant GM-32506 from the National Institutes of Health and grant NP-936B from the American Cancer Society.

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In Proof

The data referred to as C. Kocks and P. Cossart, unpublished data, is now in press: Kocks, C., Hellio, R., Gounon, P., Ohayon, H., and Cossart, P. (1993). Polarized distribution of Hysteria monocytogenes surface protein ActA at site of directional actin assembly. J. Cell Sci.. in press.