The unicellular yeast Saccharomyces cerevisiae is a model system for the establishment of cell polarity. Yeast cells proliferate by budding, which involves specialization of a small patch of the mother cell cortex and polarization of many cell constituents towards that patch, promoting growth of the bud. Genetic approaches have uncovered a large number of genes involved in these processes, yielding a working model for polarity establishment.
The poster depicts a yeast mother cell shortly before the emergence of a bud. A large number of proteins become concentrated in a polarized patch about 0.5 μm diameter. A ring of cytoskeletal filaments called septins surrounds the patch, and actin cables course through the cytoplasm, terminating near the patch. The organization of septin filaments within the ring is unknown. Although the actin cables are depicted as single filaments for simplicity, in fact each cable is thought to consist of many shorter filaments linked into parallel bundles, with each filament oriented so that the barbed (plus) end points towards the polarized patch. Myosin motors (Myo2p and Myo4p) travel along the cables towards the patch, transporting many types of cargo, including secretory vesicles, various organelles, the plus ends of cytoplasmic microtubules and RNA-protein complexes. Mitochondria are also transported along cables, though perhaps without need for myosin motors. Polarized secretion of vesicles carrying cell wall remodeling enzymes and new cell wall constituents promotes local cell wall deformation and bud emergence. Trafficking of other cargos serves to segregate organelles into the growing bud and to orient the microtubule spindle along the mother-bud axis. Not depicted are cortical actin patches, mobile short-lived structures linked to endocytosis, which are `born' near the polarized patch.FIG1
Polarization during the cell cycle
The cell cycle of budding yeast is depicted in the cell interior. Cell cycle commitment in late G1 phase triggers the assembly of the polarized patch (red) and septin ring (green), as well as the polarization of actin cables and cortical actin patches (not shown). Polarized secretion leads to bud emergence (at about the time of the G1/S transition) and then bud growth. The septin ring spreads to form an hourglass-shaped collar in the cell cortex at the neck, remaining there throughout bud growth and acting as a diffusion barrier that prevents the mixing of integral and peripheral membrane proteins between the mother cortex and the bud cortex. Early on during bud growth the polarized patch remains focused at the bud tip, but then becomes broader and dissipates as the bud enlarges. Following mitosis most of the proteins of the polarized patch reassemble at the mother-bud neck and the septin hourglass splits into two rings during cytokinesis.
Components of the polarized patch
All of the proteins known to localize within the polarized patch are depicted in the network at the center of the figure, derived using Osprey software from data curated at the Saccharomyces genome database (http://genome-www.stanford.edu/Saccharomyces/) and edited by the authors. Each protein is marked by a circle, and reported protein-protein interactions among this set are shown as connecting lines between the circles (these include reports of two-hybrid and co-immunoprecipitation data as well as direct interactions among recombinant proteins). Interacting proteins not known to localize to the patch were excluded. The proteins are divided into color-coded functional groupings.
Bud site selection
The location of the polarized patch is not random within the cell. Newborn cells carry spatial landmarks (including Bud8p at the distal tip) that influence the location of the polarized patch in the subsequent cell cycle. The Rsr1p GTPase and its guanine nucleotide exchange factor (GEF) Bud5p and GTPase-activating protein (GAP) Bud2p are required for proper localization of the polarized patch to the site specified by the spatial landmarks, and in their absence the polarized patch forms at a random location.
The Rho-family GTPase Cdc42p and its GEF Cdc24p are essential for assembly of the polarized patch and septin ring, and for the polarization of actin cables and cortical actin patches. As such, they are considered the master regulators of polarity establishment in yeast. Genetic studies indicate that the Cdc42p effectors Cla4p, Ste20p, Gic1p and Gic2p, as well as the scaffold proteins Bem1p, Boi1p and Boi2p act together with Cdc42p and Cdc24p to establish polarity.
At least four GAPs can stimulate GTP hydrolysis by Cdc42p. Phenotypic analysis suggests that Bem2p is important for polarity establishment, although it also has links to Rho1p and the control of cell wall integrity and MAPK signaling. By contrast, Rga1p, Rga2p and Bem3p have been implicated in promoting the assembly of the septin ring around the polarized patch.
The polarisome is a protein complex thought to form a link between polarity establishment factors and actin cables. The formin Bni1p promotes nucleation and growth of actin cables, and Spa2p and Bud6p are important for Bni1p localization and function. Although actin polarization is essential for bud growth, the polarisome components are not essential, perhaps because another formin, Bnr1p, is recruited to the septin ring and can nucleate polarized actin cables from there.
The type V myosin Myo2p, with associated light chains Mlc1p and Cmd1p (calmodulin), transports secretory vesicles containing the Rab-family GTPase Sec4p to the polarized patch. The SNARE-binding protein Sec1p is also polarized and essential for vesicle fusion with the plasma membrane. Myo2p also transports organelles and microtubules along actin cables, while the related Myo4p transports mRNA-protein complexes, generating mother-bud differences in protein translation.
The exocyst is a multiprotein complex that tethers secretory vesicles to the plasma membrane prior to fusion.
Glucan polymers make up a large portion of the cell wall, and the glucan synthases Fks1p and Gcs2p extrude the polymers across the plasma membrane at sites of cell growth. A cell wall protein (Cwh43p) and a putative sensor of cell wall stress (Slg1p) are also polarized, as is the Rho1p GEF Rom2p. Rho1p is a multifunctional GTPase that activates glucan synthase as well as the protein kinase C Pkc1p.
Cell wall stress activates the `cell integrity' MAPK signaling cascade, several members of which are found in the polarized patch (Rho1p, Pkc1p, Mkk1p, Mkk2p and the MAPK Slt2p). This pathway activates transcription of cell-wall-related genes and contributes to halting of the cell cycle under conditions of stress, through the morphogenesis checkpoint.
A recently identified signaling pathway termed the RAM (regulation of Ace2p activity and cellular morphogenesis) contains interacting components required for optimal polarization as well as asymmetric mother/daughter gene expression. During bud growth these proteins are localized to the polarized patch, but some components relocate to the daughter cell nucleus in the bud following mitosis.
A hierarchical model for cell polarization
The transition of a yeast cell from an unpolarized state (where only the bud site selection proteins are spatially restricted) to the polarized state depicted in the poster involves the near-simultaneous polarization of all of the structures discussed above. Examination of whether specific proteins or structures can become polarized in the absence of others has led to a hierarchical model for cell polarization. In response to a cell cycle cue, Cdc42p together with a subset of polarized patch proteins clusters into a patch at a location usually designated by the bud site selection landmarks. These proteins promote the independent assembly of the septin ring and the actin cables (and possibly also the actin patches). The septins then recruit a host of proteins to the ring, and the cables deliver more cargo, such as proteins and organelles, to the patch. Although this model accounts for most known aspects of polarization, there is probably a reinforcing cross-talk among these structures once they are polarized. For instance, the recruitment of the formin Bnr1p to the septin ring might reinforce polarized actin cable assembly, and polarized patch factors including Rho1p, Bud6p, and even Cdc42p itself might be delivered to the patch on secretory vesicles traveling along actin cables.