Cell Integrity Pathway In Budding Yeast
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Cell Integrity Pathway In Budding Yeast
Cellular integrity in the yeast Saccharomyces cerevisiae depends on the proper composition of the cell wall and is controlled by the PKC1 (Protein Kinase-C-1)-mediated signal transduction through a MAPK (Mitogen-Activated Protein Kinases) cascade. This pathway is generally activated by events that lead to a change of the cells size or shape. The pathway is also important for processes such as cell fusion, polarized growth, regulation of actin cytoskeleton polarization, control of the morphogenesis checkpoint during cell cycle and several other events that affect cell membrane or cell wall composition. The cell integrity pathway orchestrates changes in cellular morphology by controlling the expression of genes encoding enzymes involved in cell wall metabolism, by directly controlling at least one such enzyme, and by taking part in reorganizing the actin cytoskeleton. The cell integrity pathway is not a single straight cascade but rather a network of interacting signaling routes that diverge from or converge at PKC1 and the G-protein Rho1 as well as proteins controlling those. Pathways that interact with these central components of the cell integrity pathway, physically and/or genetically, are the SLT2/Mpk1 MAP kinase cascade, the calcineurin pathway, the Tor pathway, the Hog pathway, a Phosphatidylinositol pathway, CDC28-dependent control of the cell cycle, and probably additional pathways. Cell integrity pathway responds to different signals including cell cycle regulation, growth temperature, changes in external osmolarity, and mating pheromone (Ref.1).

The Mpk1 pathway is thought to be important in maintaining cellular integrity during cell wall remodeling, heat, and hypo-osmotic shock. The pathway is under the control of PKC. Signals activating the pathway are detected by sensors located at the cell surface, such as Slg1/WSC1 (Cell Wall Integrity and Stress Component-1) (also called Hsc77), WSC2 (Cell Wall Integrity and Stress Component-2), WSC3 (Cell Wall Integrity and Stress Component-3) and Mid2 . Information is then transduced via the GDP/GTP exchange factor Rom2 to the small GTPase Rho1. The latter, like all small GTPases, is considered active in its GTP-bound and inactive in its GDP-bound state. Sac7 and Lrg1 act as GAPs (GTPase-Activating Protein) for Rho1 and thus function as negative regulators. Further GAP  functions have been assigned to Bag7 and Bem2 but are less crucial for Rho1 function. Similar to many other small GTPases, Rho1 has a set of different target proteins. In its GTP-bound state it binds to and thereby activates the Beta-1,3-glucan synthase complex. It is also involved in regulation of the actin cytoskeleton by interacting with Bni1. In addition, interaction with Skn7, a regulator of oxidative stress response, has been reported. With respect to signals ensuring cellular integrity, the main effector of Rho1 is PKC1. This kinase then activates a MAP-kinase cascade consisting of the MAPKKK Bck1, the MAPKKs MKK1  and/or MKK2  and the MAPK SLT2 (Suppressor of Lyt2), also referred to as Mpk1. Rlm1 and the  SBF  complex (consisting of Swi4 and Swi6) have been reported as targets of the MAP kinase SLT2. Rlm1 is a protein of 676 amino acids with three distinct domains. The N-terminal end of the protein contains the putative DNA-binding domain, which is highly similar to the MADS (Mcm1-Agamous-Deficiens-serum response factor) box. The C-terminal part of the protein contains the transcriptional activation domain, and a central part of the protein is the target for SLT2/Mpk1-dependent phosphorylation. Rlm1 regulates transcription of a specific set of genes. Swi4 is the DNA binding subunit and transcriptional activator of  SBF  (SCB Binding Factor) and is required for normal expression of the G1 cyclin genes Cln1, Cln2, Pcl1, and Pcl2 at the G1/S transition. Swi6 is more of a regulatory subunit, because loss of Swi6 leads to constitutive intermediate levels of Cln1 and Cln2 expression. Cln1 and Cln2 are G1 cyclins that complex with the cyclin-dependent kinase CDC28 (Cell Division Cycle-28) and thereby activate the G1/S transition (Ref.2).

Tor2, Phosphatidylinositol kinase homologue, is another possible mediator of signaling to the cell integrity pathway. The Tor proteins, Tor1 and Tor2, respond to nutrient availability and activate Rom1 and Rom2, which are important components of the cell integrity pathway. Tor proteins also act as positive regulators of translation initiation and progression through G1. Tor2 has an additional function of promoting the organization of the actin cytoskeleton in G1 phase. The Beta-1,3 form of glucan is a major structural component of the yeast cell wall, and Rho1 is needed for its synthesis. The cell wall polysaccharide beta-1,3-glucan is synthesized at the cell surface by a pair of differentially expressed glucan synthases, Fks1 and Fks2. Rho1 is required in its GTP-bound form for activity of the plasma membrane-bound Fks1. The cell integrity pathway is required for cell cycle-regulated expression of Fks1 and appears to regulate the expression of Fks2, a second beta-1,3-glucan synthase gene. Rho1 therefore appears to regulate beta-1,3-glucan synthesis at two levels, direct regulation of the enzyme itself and, through the MAPK cascade, regulation of expression of the enzyme. Another mechanism by which Rho1 and the cell integrity pathway may regulate the construction of the cell wall is by regulating the delivery of another cell wall polysaccharide, beta-1,6-glucan, to the cell wall. This glucan is secreted by exocytosis and is covalently cross-linked to beta-1,3-glucan, mannoproteins, and chitin, the other major structural components of the cell wall. Cells lacking the MAPK SLT2p accumulate secretory vesicles in the bud, suggesting that the MAPK cascade is a positive regulator of exocytosis and delivery of beta-1,6-glucan to the cell surface. Rho1-GTP and other Rho  family proteins, CDC42  and Rho3, bind to Bni1, a cytoskeletal protein needed for proper bud site selection and rearrangement of the actin cytoskeleton during formation of mating projections (Ref.3).

CDC28 is a cyclin-dependent protein kinase and the master regulator of the budding yeast cell cycle. CDC28 activates PKC1 and hence the cell integrity pathway, which then mediates expression of genes encoding enzymes important for cell wall reorganization during bud emergence. The link to cell cycle control goes further, since one downstream target of the SLT2/Mpk1 protein kinase is the cell cycle-dependent transcriptional complex  SBF  with its components Swi4 and Swi6. Mating pheromone also stimulates the cell integrity pathway, and SLT2/Mpk1 phosphorylation is observed at a time when mating projections start to develop. Pheromone-dependent stimulation of the cell integrity pathway seems to involve Ste20, the PAK of the pheromone response pathway, which could indicate a direct link between these pathways. On the other hand, pheromone-induced activation of SLT2/Mpk1 phosphorylation occurs late after the first stimulation of the pheromone response pathway and seems to depend on the cell surface sensor Mid2 (Ref.4).

Other potential signaling proteins show interaction with the cell integrity pathway but seem less likely to act as upstream regulators. One such example is Bro1. Stt4 is a phosphatidylinositol 4-kinase that is proposed to act either upstream of or in parallel to the cell integrity pathway. The function of Stt4 is related to that of Mss4, a phosphatidylinositol 4-phosphate 5-kinase, and the phosphoinositide-specific PLC1. The PKC1-associated MAPK pathway of Saccharomyces cerevisiae regulates cell integrity by controlling the actin cytoskeleton and cell wall synthesis. Spatial control of cell growth in the budding yeast Saccharomyces cerevisiae involves cell wall synthesis and organization of the actin cytoskeleton. A polarized actin cytoskeleton targets secretion to the growth, or bud, site and is thus essential for the establishment and the maintenance of polarized growth. The yeast cell wall, composed of mannoproteins, Beta-1,6-glucan, Beta-1,3-glucan, and chitin, determines the cell shape and provides rigidity. Both the actin cytoskeleton and the cell wall are dynamic structures that are constantly remodeled and reorganized, in response to growth signals or environmental stress, to ensure the integrity of the yeast cell (Ref.5).