10.11 The flow of nutrients: transport and translocation
The plasma membrane is a lipoidal layer separating the aqueous ‘bubble’ of the cell from its aqueous surroundings. This separation is not complete or absolute as the cell must exchange chemicals with the environment; removing excretion products and absorbing nutrients. However, only molecules which dissolve readily in lipid are able to penetrate the membrane without assistance. Since the vast majority of molecules the cell needs to transfer across the membrane are hydrophilic rather than lipophilic, plasma membranes have evolved a range of associated transport systems that permit selective communication between the two sides of the membrane. This selectivity permits the cell to exercise considerable control over its interaction with the environment.
In an infinite solution, molecules of solute can move within the solution in two ways. Whole volumes of solution may be transported from place to place, taking solute molecules with them. This is bulk flow or mass flow and results from such things as convection flows and other large-scale disturbances within the solution. As far as living organisms are concerned, bulk flow may be achieved through cytoplasmic streaming, transpiration streams and similar processes. Although it is becoming clear that multiple motor proteins may work together to drive intracellular transport of organelles (Rai et al., 2013; Pathak & Mallik, 2017), we must emphasise that what we are describing here is different from, and additional to, the vesicle and vacuolar traffic described in Chapter 5, above (Section 5.10 The endomembrane systems; Section 5.11 Cytoskeletal systems; and Section 5.12 Molecular motors). Interestingly, strongly-bound kinesins fail to work collectively, whereas detachment-prone dyneins team up. It seems that leading dyneins in a team take short steps, while trailing dyneins take larger steps; the dyneins consequently bunch together, which shares the load more effectively and bonds the motor proteins more tenaciously to the microtubule. Even though such behaviour allows vesicle and vacuolar traffic to sustain the most rapid of extension growth rates in filamentous fungi, the bulk flow we describe here is more likely to be associated with distribution of materials (whether in solution or not) over multicellular or intercellular dimensions than with transfers via the endomembrane and cytoskeletal systems.
The second mode of solute movement is diffusion, where random thermal motion at the molecular level causes all solute molecules to move continuously. If the solution is completely homogeneous then any molecules which move out of a particular unit volume will be replaced by an identical number moving into that unit volume and the exchange of solute molecules will not be detectable. On the other hand, if there is a concentration gradient within the solution there will be a net flow of solute molecules from the high concentration end of the gradient, towards the low concentration end. Note that this gradient can be a chemical gradient of uncharged molecules (e.g. a sugar), an electrical gradient of a charged ion (e.g. K+) or a combination of the two. This diffusion process is extremely relevant to the behaviour of cells, since there is likely to be a concentration gradient across the plasma membrane for just about every solute of importance to the cell.
To traverse the biological membranes a solute must leave the aqueous phase for the lipoidal environment of the membrane, traverse that, and then re-enter the aqueous phase on the other side of the membrane. Unaided simple diffusion of molecules across biological membranes depends considerably on their solubility in lipids. There are exceptions to this generalisation, though, as some small polar molecules (such as water) enter cells more readily than would be expected from their solubility in lipid. They behave as though they are traversing the membrane by simple diffusion through gaps or pores which are transiently generated by random movements of the acyl chains of the membrane phospholipids. Transfer of these materials (like that of molecules which are soluble in the lipid bilayer of the membrane, such as O2 and CO2) depend on simple diffusion. Their rate of movement is then proportional to the concentration differential on the two sides of the membrane and the direction of movement is from the high to the low concentration side. No metabolic energy is expended and no specific membrane structures are involved in this mode of transfer, but net transfer ceases when the transmembrane concentrations equalise.
Only a minority of compounds pass through biological membranes in vivo by simple diffusion; the vast majority of metabolites that the cell needs to absorb or excrete are too polar to dissolve readily in lipid and too large in molecular size to make use of transient pores. To cope with these circumstances the membrane is equipped with solute transport systems. This applies to intracellular membranes bounding compartments within the cell as well as to the plasma membrane. The essential component of any transport system is a transporter molecule, a protein which spans the membrane and assists transfer of the metabolite across the lipid environment of the membrane.
With both passive and active transporters, substrate translocation depends on a conformational change in the transporter such that the substrate binding site is alternately presented to the two faces of the membrane. These transporters are transmembrane glycoproteins of around 500 amino acids arranged into three major domains: 12 α-helices spanning the membrane, a highly charged cytoplasmic domain between helices 6 and 7, and a smaller external domain, between helices 1 and 2, which bears the carbohydrate moiety. Sequence homology between the N- and C-terminal halves of the protein suggests that the 12 α-helix structure has arisen by the duplication of a gene encoding a 6-helix structure. Ion channels are different as their polypeptide subunits form a β-barrel containing a pore. One loop of the polypeptide is folded into the barrel and amino acids of this loop determine the size and ion selectivity of the channel. This transporter alternates between open and closed conformations.
If the transfer is passive with no requirement for metabolic energy then the transport process is described as facilitated diffusion. Such a process still depends upon a concentration differential existing between the two sides of the membrane, transfer occurring ‘down the gradient’ (towards the compartment which has the lower concentration). However, transfer is much faster than would be predicted from the solubility of the metabolite in lipid, the high rate of transfer depending on the fact that the transporter and the transporter/metabolite complex are highly mobile in the lipid environment of the membrane. The major differences from simple diffusion are that facilitated diffusion exhibits:
- high substrate specificity;
- saturation kinetics.
Showing saturation kinetics means that as the concentration of the metabolite being transported is increased, the rate of transport increases asymptotically towards a theoretical maximum value at which all the transporters are complexed with the metabolite being transported (i.e. transporters are saturated).
Facilitated diffusion can transport a specific substrate very rapidly; but can only equalise the concentrations of the transported metabolite on the two sides of the membrane. Yet in many cases the cell needs to transfer a metabolite against its concentration gradient. The prime example will be where the cell is absorbing a nutrient available at only a low concentration; if growth of the cell is not to be limited by the external concentration of the nutrient, the cell must be able to accumulate the nutrient to concentrations greater than those existing outside. In which case an adverse gradient of concentration will have to be established and maintained. Neither simple diffusion nor facilitated diffusion can do this; to achieve it the cell must expend energy to drive the transport mechanism. Such a process is called active transport.
Active transport is a transporter-mediated process in which movement of the transporter/substrate complex across the membrane is energy dependent. The transporter exhibits the same properties as a facilitated transport transporter (saturation kinetics, substrate specificity, sensitivity to metabolic inhibitors). In addition to these properties, active transport processes characteristically transfer substrate across the membrane against a chemical and/or electrochemical gradient, and are subject to inhibition by conditions or chemicals which inhibit metabolic energy generation.
The mechanism is often a co-transport in which the movement of an ion down its electrochemical gradient is coupled to transport of another molecule against its concentration gradient. When the ion and the transported substrate move in the same direction the co-transporter is called a symport, whereas transporters which transport the two in opposite directions are termed antiporters. The electrochemical gradients, most usually of protons or K+ in fungi, are created by ion pumps in which hydrolysis of ATP phosphorylates a cytoplasmic domain of the ion channel. Consequential conformational rearrangement of the protein then translocates the ion across the membrane and reduces the affinity of the binding site to release the ion at the opposite membrane face. Dephosphorylation restores the pump to its active conformation (and may translocate another ion or molecule in the opposite direction).
Complex interactions occur in transport of anions, cations and non-electrolytes; interactions which may depend on metabolic, chemical, biophysical and/or electrochemical relationships between a number of different molecular species and with the rest of metabolism. There are indications of what might be called transport strategy in operation in most cells. Single uptake systems are rarely encountered; dual or multiple systems are the norm, the different components being suited to different environmental conditions the organism may encounter. Multiple uptake systems inevitably result in complex uptake kinetics which might be indicative of physically separate transport transporters, each showing Michaelis-Menten kinetics (like the glucose transporters in Neurospora), or of single molecules exhibiting kinetics modulated by their environment (like the glucose transporter in Coprinopsis; Moore & Devadatham, 1979; Taj Aldeen & Moore, 1982).
Whatever the physical basis, the regulatory properties of the components of such ‘families’ of transport processes appear to be interlinked to ensure that nutrient uptake is maintained at a reliable level whatever the variation in substrate availability in the environment. Probably the most important generalisation that can be made about transport processes, though, is that for almost all of them the active extrusion of protons from the fungal cell seems to be essential. The proton gradient so established provides for uptake of sugars, amino acids and other nutrients by proton co-transport down the gradient, and is directly involved in cation transport like the K+/H+ exchange or antiport. So, each fungus possesses multiple uptake systems for most nutrients but the same basic process (active H+ extrusion) energising most if not all.
A crucial point, which has not yet been taken into account, is that the transport systems so far described will inevitably alter the solute concentrations of the cell and thereby influence the movement of that all-pervading nutrient, water. Water is a significant (even if often overlooked) component of innumerable biochemical processes. For example, every hydrolytic enzyme reaction uses a molecule of water, every condensation reaction produces a molecule of water and respiration of 1 g of glucose produces 0.6 g of water. The water relationships of the fungal cell are an important aspect of its overall economy. Water availability is determined by its potential energy; referred to as the water potential, symbolised by the Greek letter psi (Ψ). Zero water potential is the potential energy of a reference volume of free, pure water. The water in and around living fungal cells will have positive or negative potential energy relative to that reference state, depending on the effect(s) of osmotic, turgor, matrix and gravitational forces. Water will flow spontaneously along a water potential gradient, from high to low potentials, though in the normal state for most fungi this will mean from a negative to a more negative potential. The lower the water potential the less available is the water for physiological purposes and the greater is the amount of energy that must be expended to make the water available.
On the face of it, two things need to be considered. One is some sort of compensation for change in the solute relationships of the cell resulting from uptake of some substrate; such a process would further reduce the potential of the cell water and increase the tendency of external water to influx. The other is to provide the cell with a means to regulate its water uptake even though the external water potential is uncontrollable. In fact, of course, these are just two facets of the same problem. In either case the fungus must cope with water potential stress and the evidence indicates that solute transport systems provide the mechanism which permits this. The internal maintenance of turgor pressure by movement of water across the membrane is related to transport of ions across the membrane and to the breakdown of macromolecules and biosynthesis of solutes. Inorganic ions usually make the greatest percentage contribution to the osmotic potential of the protoplasm. The main ions involved are K+ and Na+, with Cl- being moved to balance the cation content. Some organic solutes also make major contributions, including glycerol, mannitol, inositol, sucrose, urea and proline.
The most immediate response to water potential stress is change in cell volume by the rapid flow of water into or out of the cell. The consequent change in turgor affects the cell membrane permeability and electrical properties so that the cell can restore the volume by transporting ions or other solutes across the membrane and/or by synthesising solutes or by obtaining them by degrading macromolecules. Response to water potential stress can be extremely rapid. Experimentally this is particularly evident in fungal protoplasts, the size of which alters soon after change in the solute concentration of the suspending medium. Such behaviour attests to the ready permeability of the cell membrane to water.
Polar water molecules can move across cell membranes despite their lipid (hydrophobic) environment forming a natural barrier to their transport; such transport is called osmosis, which is just a special type of simple diffusion. Diffusion of water through lipid sounds like a very unlikely event, and it is, but what drives it is what drives all diffusion events, which is the relative concentrations of the diffusing molecule at the ‘source’ and at the ‘sink’. In the case of water molecules penetrating a lipid membrane, the concentration of water in the lipid phase (the ‘sink’) will be extremely low (the solubility of water in lipid is about 1 molecule of water per million molecules of lipid), but the concentration in the aqueous phase (the ‘source’) will be extremely high [we’ll leave you to calculate the concentration of water; remember a molar solution contains the gram-molecular mass of a solute in one litre of solution, and that the mass of a litre of water, by definition, is 1,000 g and the molecular mass of H2O is 18. If you think all that’s too easy, Google Avogadro’s constant and work out how many water molecules there are in a 200 ml glass of water].
Such extreme diffusion gradients, with the additional facts that the water molecule is very small and the surface area to volume ratio of the cell is large, offset the very low permeability of the membrane and allow water to diffuse through the lipid bilayer. That’s not the whole story, of course, because in some membranes the water flux is too high to be accounted for by simple diffusion alone. In such cases, water migrates by facilitated diffusion (see Section 5.13) through pores or channels provided by proteins called aquaporins that form membrane-spanning complexes. Water moves through these channels passively in response to osmotic gradients (Nehls & Dietz, 2014).
The managed flow of water, coordinated with control of the wall synthetic apparatus, must be a prime factor in controlling the inflation of fungal cells which is responsible for many of the changes in cell shape which characterise fungal cell differentiation. Turgor also contributes to flow along the fungal hypha. As this is a filamentous structure, flow of water and solutes within the hypha (i.e. translocation) is of enormous importance. Although our current view of apical growth requires that fungi can organise rapid translocation and specific delivery of various vacuoles and microvesicles, more general water flow along the hypha is driven by a turgor gradient and solutes are translocated by this turgor-driven bulk flow. Translocation of nutrients of all sorts in this manner is of crucial importance to morphogenesis because it must be the main way in which developing multicellular structures, such as a fruit body developing on a vegetative colony, are supplied with nutrients and water. Translocation is ably discussed by Jennings (2008; see his Chapter 14) and the mechanism can best be illustrated by quoting his description of the way in which Serpula lacrymans (the major timber decay organism in buildings in northern Europe) translocates carbohydrate:
“Mycelium attacks the cellulose in the wood, producing glucose, which is taken into the hyphae by active transport. Inside the hypha, glucose is converted to trehalose, which is the major carbohydrate translocated. The accumulation of trehalose leads to the hypha having a water potential lower than outside. There is a flux of water into the hyphae and the hydrostatic pressure so generated drives the solution through the mycelium. The sink for translocated material is the new protoplasm and wall material produced at the extending mycelial front. The mechanism of translocation in S. lacrymans is thus the same as that now accepted for translocation in the phloem of higher plants, namely osmotically driven mass flow. (Jennings, 2008; p. 459).”
By measuring the increase in volume of developing cord networks of Phanerochaete velutina, Heaton et al. (2010) established that hyphal (and cord) growth induces mass flows across the whole mycelial network. This compensates for the fact that osmotically driven water uptake is often distal from the tips, and results in a rapid global response to local fluid movements. Coupling of growth and mass flow enables development of efficient, and highly adaptive mycelial transport networks. Velocity of fluid flow in each cord becomes a local signal that conveys information about the role of each cord within the mycelium; cords carrying fast-moving or large fluid flows were significantly more likely to increase in size than cords with slow-moving or small currents.
Jennings' (2008) description quoted above could be paraphrased to apply to other circumstances by, for example, featuring nutrients other than carbohydrates and/or alternative sinks, such as fruit bodies or particular tissues in fruit bodies. Importantly, this bulk flow does not have to be unidirectional within a tissue. Because the tissue is comprised of a community of hyphae, different hyphae in that community may be translocating in different directions simultaneously. Nutrients labelled with different radioisotopes have been used to demonstrate that carbon is translocated simultaneously in both directions along rhizomorphs of Armillaria mellea. In mycorrhizas, carbon sources from the host plant and phosphorus absorbed by the hyphae from the soil must move simultaneously in opposite directions. Indeed, the flow of carbon in mycorrhizas must be fairly complex as carbon can be transferred between two different plants which are connected to the same mycorrhizal system. Although much remains to be learned, there is clear evidence that nutrients (including water in that category) can be delivered over long distances through mycorrhizal hyphal systems, and that the flows can be managed and targeted to specific destinations in this ‘mycorrhizal transportome’ as circumstances demand (Courty et al., 2016).
Long distance translocation (up to 50 cm) of a 14C-radiolabelled metabolically inert amino acid analogue, α-amino-isobutyric acid (14C-AIB) has been demonstrated in the vegetative mycelium of Agaricus bisporus (Herman et al., 2020). Translocation to the periphery of the mycelium was observed in actively growing vegetative mycelium with a velocity of up to 6.6 mm h‑1, which was 30 × greater than the growth rate. The 14C-AIB was also translocated between colonies joined by hyphal fusions in a manner depending on the relative growth rates of the colonies, and was redirected to sporophores when they started to develop, especially via cords which could show a 5 × increased rate of translocation.
Updated January, 2021
