Maintenance of the cell wall integrity in S. cerevisiae

The yeast CWI pathway becomes activated during the requirement of cell wall remodeling, such as vegetative growth or under stress conditions which challenge the cellular integrity. Such stress conditions involve physical damage, temperature shifts, a hypo-osmotic shock or interference with antifungal agents ¹⁴⁰. In theory, cell wall stress is detected by sensors in the cell wall. Upon activation, these sensors bind by their cytoplasmic tail to the GDP/GTP exchange factor Rom2, which in turn activates the small GTPase Rho1 (Fig. 12). Rho1 is considered as a master regulator for the CWI pathway since it is also involved in cell wall synthesis, cell surface- as well as cell cycle signaling ¹⁴⁰. Moreover, Rho1 interacts with the protein kinase C (Pkc1), which activates the MAPK cascade ¹⁴¹. The MAPK cascade is composed of the MAP kinases Bck1, Mkk1, Mkk2 and Slt2 (also known as Mpk1) and triggered by downstream phosphorylation. Slt2 is mostly present in the nucleus but also occurs in the cytoplasm upon cell wall stress ¹⁴². In the nucleus, Slt2 activates the transcription factor Rlm1 and the heterodimeric SBF complex subunit, composed of Swi4 and Swi6, which are involved in the regulation of cell wall synthesis and cell cycle control ⁸⁷.

1.5.2. Cell wall integrity sensor proteins

A group of five sensor proteins has been described for the CWI pathway in S. cerevisiae, which can be separated into the Wsc-type family with three members (Wsc1, Wsc2 and Wsc3) ^86,139,143 and the Mid-type family with two members (Mid2 and Mtl1) ^144,145. A similar protein, Wsc4, is not a CWI sensor, as it resides in the ER membrane and is probably involved in protein translocation ¹⁴⁶. These CWI sensor proteins share a common domain structure (Fig. 13).

An N-terminal secretion signal mediates their transport to the cell membrane. In addition, a single transmembrane domain (TMD) anchors them in the cell membrane.

These domains possess a cytoplasmic, unstructured tail of various lengths, which enables the interaction with downstream components of the CWI pathway ¹⁴⁷. The tail contains a putative Rom2-binding site, as well as several putative phosphorylation sites.

Moreover, an extracellular serine/threonine-rich region (STR), variable in length, builds a spring-like structure, reaching into the cell wall. This region is highly mannosylated, which is thought to stabilize the rod-like shape of the spring and which is necessary for its sensor function ¹⁴⁸. It shows the physical properties of a nanospring, depending on the degree of glycosylation ¹⁴⁹. The main difference between the two CWI sensor families is the presence of an N-terminal, conserved and cysteine-rich domain (CRD) in

Fig. 13 The CWI sensors Wsc1-3, Mid2, Mtl1 and their proposed structural organization in the S. cerevisiae cell wall ¹³⁹.

conserved cysteine-residues by a molecular weight of about 10 kDa, indicating a particularly rigid structure. Instead, the two Mid-type sensors carry an N-glycosylated asparagine residue at the head-group position, which has been reported to be required for their sensor function ^87,150. The CRD has been shown to be involved in clustering of the Wsc sensors ¹⁵¹. It has features of a lectin binding domain and is supposed to be in contact with cell wall β-glycans ¹⁵². In general, CRDs occur in proteins with diverse functions, including dimerization and DNA-interaction ⁸⁶.

It is believed that the CWI sensors have a mechanoreceptor function: in theory, the CRD anchors within the cell wall and is connected to the TMD via the STR region.

When the cell wall is under mechanical stress, the CRD becomes dislocated in relation to the TMD. As a consequence, tension is exerted on the intermediate spring-like STR and it becomes expanded. This might trigger a conformational change in the cytoplasmic tail, which enables interaction with Rom2 and thus induces a stress response ¹⁵¹. This theory is supported by an atomic force microscopy study, in which Wsc1 showed the dynamic behavior of a linear hookean spring ¹⁴⁹. The different length in the STR might then be explained by the ability of stress detection in different layers of the cell wall, since different stress factors might affect different cell wall layers by varying degrees ⁸⁷.

The deletion of WSC1 leads to hypersensitivity to stress conditions including temperature shifts and the addition of cell wall destabilizing compounds, such as calcofluor white or congo red and the defect is enhanced by the additional deletion of WSC2 and WSC3 ^86,143,153. In addition, Wsc1-GFP localized at sites of polarized cell growth, like bud-necks and tips of emerging buds, whereas Mid2 has been shown to have a more homogeneous distribution and seems to be related to a general stress response and mating ¹⁵³. Mtl1 is supposed to be involved in the stress response upon glucose starvation and oxidative stress ^154,155. Furthermore, the deletion of MLT1 leads to temperature sensitivity in combination with mid2∆ ¹⁴⁵. The sensors Wsc1 and Mid2 have been suggested to be mainly responsible for CWI signaling with maybe partly overlapping functions, although Mid2 has no CRD ¹⁵².

1.5.3. The Wsc1 cysteine-rich domain

It has been shown by HEINISCH et al., that Wsc1 builds membrane clusters (Fig. 12) or

‘microdomains’ in vivo with a diameter of about 200 nm, increasing in size under stress conditions ¹⁵¹. The microdomains of Wsc1 indicate a moderate overlap with other Wsc-type sensor clusters, but not with those of the Mid-Wsc-type sensors or other established

clustering and signaling, since the exchange of cysteines to alanine within in the Wsc1-CRD resulted in an even Wsc1-distribution and a sensitivity towards cell wall stress, comparable to that of a wsc1∆ strain ¹⁵¹. In line with this, the addition of dithiothreitol as a reducing agent for disulfides leads to a similar result in cells with the wild type Wsc1 ¹⁵⁷. In general, clustering of membrane sensors can enhance the cytoplasmic signal strength or might play a role in a threshold limiting for signal generation ⁸⁷. Since Wsc1 is expected to bind Rom2 by its cytoplasmic part, clustering has been suggested to form a ‘Wsc1 sensosome’ signaling complex ¹⁵¹. However, the specific mechanism of the Wsc clustering and its molecular triggers remain unknown.

As mentioned previously, the presence of eight highly conserved cysteines, indicating the formation of four disulfide bridges, is a particularly large amount for a small protein-like the Wsc-CRDs. These cysteines are found in the Wsc family among different yeast species and in homologous domains of other species. Currently, there are 577 proteins with Wsc-domains provided by the SMART database ¹⁵⁸. The fungus Trichoderma harzianum has a Wsc domain with an exoglucanase (ThCRD2) function, which could indicate a carbohydrate binding function for the Wsc family of S. cerevisiae.as well. Moreover, the closely related yeast Kluyveromyces lactis has cell wall sensors with Wsc-domains, which are also involved in cell wall integrity ¹⁵⁹. In the more distantly related yeast Schizosaccharomyces pombe, Wsc1 homologs have also been identified, but they did not trigger the CWI pathway ¹⁶⁰. The human protein Krm1 has a Wsc-domain as well, which structure has been solved recently ¹⁶¹.

Krm1 is a transmembrane receptor, found in a ternary complex of the Wnt signaling pathway, which is important during embryonic development and tissue homeostasis ¹⁶². The Wsc domain of Krm1 has been shown to be involved in binding of the Wnt antagonist Dkk1 ¹⁶¹. Yet another Wsc domain is found in the mammalian protein Polycystin-1 (Pdk1) ¹⁶³. It has been shown to play an essential role in renal tubular morphogenesis, and malfunction causes cystogenesis in human autosomal-dominant polycystic kidney disease. Moreover, Pdk1 is also considered as an ion-channel regulator or to be involved in protein-protein and protein-carbohydrate interactions ¹⁶³. The specific function of the Wsc-domain in Pdk1 remains unknown.