21st Century Guidebook to Fungi, SECOND EDITION, by  David Moore, Geoffrey D. Robson and Anthony P. J. Trinci

Table of Contents

1. Chapter 4: Hyphal cell biology and growth on solid substrates
2. Chapter 5: Fungal cell biology
   1. Mechanisms of mycelial growth
   2. The fungus as a model eukaryote
   3. The essentials of cell structure
   4. Subcellular components of eukaryotic cells
   5. The nucleus
   6. The nucleolus and nuclear import and export
   7. Nuclear genetics
   8. Mitotic nuclear division
   9. Meiotic nuclear division
   10. mRNA translation and protein sorting
   11. The endomembrane systems
   12. Cytoskeletal systems
   13. Molecular motors
   14. Plasma membrane and signalling pathways
   15. Fungal cell wall
   16. Cell biology of the hyphal apex
   17. Hyphal fusions and mycelial interconnections
   18. Cytokinesis and septation
   19. Yeast-mycelial dimorphism
   20. Chapter 5 References and further reading about the cell cycle
   21. Buy a PDF of Chapter 5
3. Chapter 6: Structure and synthesis of fungal cell walls

5.14 Plasma membrane and signalling pathways

The prime function of the membranes of fungi, like those of other eukaryotic cells, is to provide a barrier between the cell and its environment. Plasma membranes are composed of a phospholipid bilayer, but this is not a static barrier. An enormous number and variety of proteins are anchored into the membrane, which carry out a tremendous range of functions. Sterols are also a critical component of the fungal membrane and serve to regulate membrane fluidity and the activity of membrane-associated enzymes and transport mechanisms. In plants the most common sterols are sigmasterol, sitosterol and campesterol. In animal cells and Oomycota, cholesterol is the chief sterol in the membrane, whilst in the majority of fungi the major sterol is ergosterol (the exceptions being the Chytridiomycota, where the dominant sterol is again cholesterol). This difference in the primary sterol component of fungal and mammalian cells prompted development of two classes of antifungal agents, the polyenes and the azoles (Robson, 1999).

The polyene antifungals, which include nystatin and amphotericin, bind by hydrophobic interaction to the ergosterol component of the membrane forming pores leading to a loss of plasma membrane integrity. The azole antifungals, which include the triazoles and imidazoles, act primarily by inhibiting an enzyme, sterol 14-demethylase, which is involved in ergosterol biosynthesis, resulting in ergosterol depletion in the membrane and the accumulation of 14-O’-methylsterols; these changes in composition result in changes in membrane fluidity that adversely affect transport processes and wall biosynthesis and ultimately result in death of the fungus (see Chapter 18).

In order for fungi to grow, external nutrients must be assimilated across the plasma membrane. To absorb nutrients successfully from the surrounding environment, the fungi possess a diverse range of specific transport proteins in the plasma membrane (van Dijck et al., 2017). Three main classes of nutrient transport occur in fungi, facilitated diffusion, active transport and ion channels. Fungi usually contain two transport mechanisms for the assimilation of nutrients such as sugars and amino acids. One is a constitutive low affinity transport system which allows the accumulation of a nutrient when it is present at a high concentration outside the hypha. This process of facilitated diffusion is not energy dependent and does not allow accumulation of solutes against a concentration gradient. However, the ‘facilitator’, more often called the permease, carrier or transporter, is a polypeptide that confers enzyme-like specificity to the uptake process.

When the external solute concentration is lower than it is within the cell (which is probably more often the case in the natural environment), a second class of carrier protein is induced that has a higher affinity for the nutrient and can take up the solute against the concentration gradient; this, though, is energy-dependent, occurring at the expense of ATP, and is called an active transport process. Most active transport processes in the fungal cell are powered by an electrochemical proton gradient that fungi create by pumping hydrogen ions out of the hyphae, at the cost of ATP hydrolysis, using proton pumping ATPases in the plasma membrane. The resultant proton gradient provides the electrochemical gradient which can drive nutrient uptake by a carrier that couples nutrient uptake to the flow of hydrogen ions back down the gradient. As active transport processes are usually induced by environmental conditions (often low nutrient concentration), fungi are evidently capable of adapting their transport mechanisms according to the external solute concentration; this assures continued nutrient supply over a variety of environmental circumstances.

Ion channels are highly regulated pores in the membrane which allow influx of specific ions into the cell when open. A number of ion channels have been described in fungi by patch-clamp electrophysiology experiments analogous to studies conducted with mammalian cells. Patch clamping involves measuring the current flowing across a patch of the plasma membrane, which can be used to study the flow of particular ions across the membrane. In fungi, the cell wall must be removed first by incubating mycelium in an osmotic stabiliser and a mixture of lytic enzymes which digest away the cell wall. This produces naked sphaeroplasts (which are mostly wall-free) or protoplasts (which are entirely wall-free) from which patches of membrane can be removed with micropipette electrodes. Several different channel types have been identified, including anion selective channels (such as Cl^-) as well as various cation-selective channels, especially K⁺ and Ca²⁺ permeable channels.

Channels that carry an inward flux of K⁺ ions are thought to be involved in regulating the internal turgor pressure of the hypha (the bulk of the osmotic potential of the hypha is provided by inorganic ions most of the time). The presence of a mechanosensitive or stretch-activated Ca²⁺ channels has attracted interest. These are opened when the membrane is under mechanical stress and may play important roles in the generation of the calcium gradients in response to physical changes to the membrane, which might be caused, say, by rapid water fluxes in the cell (rapid water influx stretching the membrane/rapid water loss releasing tension), or by physical pressure (encountering an obstacle, gravitational stress, etc.). This, of course, amounts to an environmental sensory system since the mechanosensitive ion flux can be linked to any of a range of response systems.

The hypha must also be able to detect the nutrient status of the substratum over, or through, which it is growing. This is the responsibility of signal transduction pathways which recognise a particular chemical at the plasma membrane/outside world interface and then convey the information into the cell. Intermediary metabolism is obviously crucial to fungal growth and development (Moore, 1998, 2000) and imposes numerous regulatory events on the cell. Primary metabolites like glucose are especially important, and because the amount of available glucose (for example) can fluctuate wildly in the heterogeneous substrata that fungi inhabit, individual fungal hyphae must be able to sense the amount available to them and respond appropriately.

When readily utilisable sugars, such as glucose or fructose, are added to fungi which are starved (= derepressed) for carbohydrates, a wide range of metabolic responses follow rapidly, some of which are mediated by a transient rise in the levels of cyclic-AMP (cAMP), this being the final product in the cycle of ATP dephosphorylation. These responses include:

• inhibition of gluconeogenesis (generation of glucose from non-sugar carbon substrates like pyruvate, and glucogenic amino acids, like alanine and glutamate);
• activation of glycolysis (to amplify energy generation from whatever sugar and non-sugar carbon substrates may be available);
• activation of trehalase, which can produce two molecules of glucose by hydrolysing one molecule of the disaccharide trehalose (the characteristic storage sugar of fungi).

The immediate response to external nutrient supply level will be to modify the activity of existing enzyme systems (as described above), the ultimate effect of (for example, external glucose) is control of gene expression. Glucose has two major effects on gene expression in Saccharomyces cerevisiae: it represses expression of many genes, including those encoding proteins in the respiratory pathway (including cytochromes) and enzymes for metabolism of alternative carbon sources (e.g. galactose, sucrose, maltose); it also induces expression of genes required for glucose utilisation, including genes encoding glycolytic enzymes and glucose transporters.

There are two signal transduction pathways responsible for these effects of glucose on yeast. The first uses the Mig1 transcriptional repressor, which is a zinc finger transcription factor whose function is inhibited by the Snf1 protein kinase. The Snf1 protein kinase is a glucose sensor in the plasma membrane, and is an AMP-activated serine/threonine protein kinase required for transcription of several glucose-repressed genes; the Snf1 complex phosphorylates Mig1 in response to glucose and thereby releases the transcriptional repression of those genes for which Mig1 is responsible (Papamichos-Chronakis et al., 2004).

The signalling pathway responsible for glucose induction centres on the Rgt1 transcription factor which regulates expression of several glucose transporter genes in response to glucose. It binds to promoters and acts as a transcription activator (Kayikci & Nielsen, 2015). In the absence of glucose, Rgt1 function is inhibited by the SCF protein complex. The SCF complexes are a family of ubiquitin ligases that target specific proteins for destruction at the 26S-proteasome (Willems et al., 2004).

Many of these responses are mediated by the activation of cAMP-dependent protein kinases modifying the activity of existing proteins by phosphorylation. The increase in cAMP is due to the activation of the membrane-bound enzyme adenylate cyclase as a result of the activation of a class of small GTP-binding proteins, the RAS proteins. RAS proteins are binary switches, cycling between ON and OFF states during signal transduction. Activation of RAS leads to the exchange of RAS-bound GDP for GTP, causing a conformational change that leads to stimulation of adenylate cyclase activity. The RAS protein complex includes an intrinsic GTPase, which converts GTP-bound RAS to GDP-bound RAS, thus returning RAS to its resting state in the absence of an activator. The evidence strongly suggests that the RAS pathway forms part of a global mechanism for general signalling that control processes as diverse as cytoskeletal integrity and cell-cell fusion and exocytosis. RAS signalling at the plasma membrane of Aspergillus hyphal tips is involved in the polarisation of the actin cytoskeleton that is required for hyphal growth and, possibly, for asexual morphogenesis (Harispe et al., 2008; Noble et al., 2016). We should also add that defective RAS proteins are the cause of several human diseases (Simanshu et al., 2017).

RAS is a G-protein: a regulatory GTP hydrolase that cycles between the two conformations we’ve described, activated (RAS-GTP) or inactivated (RAS-GDP). RAS is attached to the cell membrane by a chemical modification called prenylation, which is the addition of hydrophobic molecules to a protein. Protein prenylation involves the transfer of either a farnesyl or a geranyl-geranyl group to C-terminal cysteine(s) of the protein by farnesyltransferase or geranylgeranyltransferase. Prenyl groups enable proteins to attach to cell membranes, serving as a lipid anchor for the protein (Wang & Casey, 2016).

Throughout the eukaryotes many hormones, neurotransmitters, chemokines (proteins secreted by cells to control other cells), mediators (local mediators are membrane-modifying lipids, such as omega-3-fatty acids, but mediator is also a large protein complex that interacts with the RNA polymerase II machinery), and sensory stimuli exert their effects on cells by binding to G-protein-coupled receptors. More than a thousand such receptors are known, and more are being discovered all the time. Heterotrimeric G-proteins (made up of a, b, and g subunits) transduce ligand binding to these receptors into intracellular responses. There are four main classes of G-proteins:

• Gs, which activates adenylyl cyclase;
• Gi, which inhibits adenylyl cyclase;
• Gq, which activates phospholipase C;
• G-proteins of unknown function.

G-proteins are inactive in the GDP-bound state and are activated by receptor-catalysed guanine nucleotide exchange resulting in GTP binding to the Ga subunit, which leads to dissociation of Gb and Gg subunits that activate downstream effectors. Since the first description of G proteins in yeasts and filamentous fungi in the 1990s, they have been shown to be essential for growth, asexual and sexual development, and yeast-hyphal dimorphism and virulence of both animal and plant pathogenic species (Li et al., 2007; Shi et al., 2007).

G-protein and RAS phosphorylation also initiate the MAP kinase signalling pathway. An extracellular factor affixes to a receptor on the outer membrane surface, the resultant conformational change in the receptor dimer causes it to become phosphorylated and it is then able to activate a specific G-protein, which in turn catalyses the phosphorylation of RAS by GTP into its active form. In this form the RAS is able to catalyse the phosphorylation of the beginnings of the MAP kinase cascade. This cascade begins with MAP Kinase Kinase Kinase (MAPKKK), a family of enzymes which in turn activates the second step in the cascade, the family of MAP Kinase Kinases (MAPKK), which then activate the MAP Kinases (MAPK) which have the actual effects of stimulating the transcription of specific genes in the nucleus. MAP kinase pathways are remarkably conserved throughout evolution, functioning as key signal transduction components in fungi, plants, and mammals. ‘MAPK’ stands for ‘Mitogen Activated Protein Kinase’ so the genes that are particularly controlled are those required for nuclear division and cell differentiation. MAPKs produce many of the responses that are induced in cells by changes in environmental conditions and/or exposure to external stimuli. These pathways were first revealed in budding yeast in which pathways includes activation by pheromones during formation of mating projections in shmoos (see Section 5.9; CLICK HERE to see it now) (Chen & Thorner, 2007); and a range of other cellular events in filamentous fungi (Erental et al., 2008; Read et al., 2009). Of particular interest is that in plant pathogenic fungi the MAPK cascades in the two organisms mutually contribute to an interconnected molecular dialogue between plant and fungus. Fungal MAPKs promote penetration of host tissues, while plant MAPKs activate plant defences. However, some pathogenesis-related processes controlled by fungal MAPKs lead to the activation of plant MAPK cascade signalling. Conversely, plant MAPKs that promote defence mechanisms against fungal cells, lead to a fungal protective stress responses mediated by fungal MAPK cascades (Hamel et al., 2012).

Although there are differences in molecular details, signalling pathways triggered by G-protein-linked receptors have several points in common and all have the essential similarity that their function is to make a massive response to a very small signal. The ‘cascade’ structure of the pathway enables this signal amplification; each step produces molecules that can modify even more molecules in the next step. Consequently, a single extracellular signal molecule can cause many thousands of intracellular protein molecules to be altered. 

We have mentioned protein prenylation as a means of directing specific proteins to membranes [CLICK HERE to see it now]. A related process applied to many cell-surface proteins is to attach glycosylphosphatidylinositol (GPI) as a lipid anchor. The GPI anchor is a post-translational modification of proteins with a glycolipid carried out in the ER; the carboxy-terminal signal sequence of the preprotein remains in the ER membrane with the rest of the protein in the ER lumen. Final removal of the signal peptide is combined with attachment of the finished protein’s new carboxy-terminus to an amino group on a pre-assembled GPI precursor located in the inner leaflet of the ER. Depending on the organism, cell type, and protein, the GPI backbone can be chemically modified in several ways.

The anchored protein can then be delivered to the plasma membrane in a vesicle and located with the GPI anchor in the outer leaflet of the plasma membrane and the protein on the cell exterior. GPI anchors are used ubiquitously in eukaryotes and most likely in some Archaea, but not in Eubacteria. GPI-anchored proteins are the major form of cell-surface proteins in protozoa. In fungi, many GPI-anchored proteins are ultimately incorporated into the cell wall; their GPI-anchor is partially trimmed off at the plasma-membrane just before their incorporation into the cell wall, though the glycan part of the GPI-anchor remains and is linked to the cell wall glucans. In humans, there are at least 150 GPI-anchored proteins, with a variety of roles: receptors, adhesion molecules, enzymes, transcellular transport receptors and transporters, and protease inhibitors (Kinoshita, 2016).

Updated July, 2019