Pascal Ribéreau-Gayon - Handbook of Enology - Volume 1

The plasma membrane also produces cell wall glucan and chitin. Two membrane enzymes are involved: β ‐1,3‐glucan synthase and chitin synthase. These two enzymes catalyze the polymerization of glucose and N ‐acetyl‐glucosamine, derived from their activated forms (uridine diphosphate or UDP). Mannoproteins are essentially produced in the endoplasmic reticulum (ER; Section 1.4.2). They are then transported by vesicles that fuse with the plasma membrane and deposit their contents at the exterior of the membrane.

Finally, certain membrane proteins act as specific cell receptors. Therefore, the yeast can react to various external stimuli such as sexual hormones or changes in the concentration of external nutrients. The activation of these membrane proteins triggers the release of compounds such as cyclic adenosine monophosphate (cAMP) into the cytoplasm. These compounds serve as secondary messengers that set off other intercellular reactions. The consequences of these cell mechanisms in the alcoholic fermentation process merit further study.

Bounded by the plasma membrane and the nuclear membrane, the cytoplasm contains a basic cytoplasmic substance or cytosol. The organelles (ER, Golgi apparatus, vacuole, and mitochondria) are isolated from the cytosol by membranes.

Cytosol is a buffered solution, with a pH between 5 and 6, containing soluble enzymes,glycogen, and ribosomes.

Glycolysis and alcoholic fermentation enzymes ( Sections 2.2.1and 2.2.2) as well as trehalase (an enzyme catalyzing the hydrolysis of trehalose) are present. Trehalose is a reserve disaccharide, also cytoplasmic, that ensures yeast viability during the dehydration and rehydration phases by maintaining membrane integrity.

The lag phase, preceding the log growth phase in a sugar‐containing medium, is marked by a rapid breakdown of trehalose, linked to an increase in trehalase activity. This activity is itself closely related to an increase in the amount of cAMP in the cytoplasm. This compound is produced by a membrane enzyme, adenylate cyclase, in response to the stimulation of a membrane receptor by an environmental factor.

Glycogen is the principal yeast carbohydrate reserve substance. Similar in structure to animal glycogen, it accumulates during the stationary phase in the form of spherical granules of about 40 μm in diameter.

When observed under the electron microscope, the yeast cytoplasm appears rich in ribosomes. These tiny granulations, made up of ribonucleic acids and proteins, are the center of protein synthesis. Associated with polysomes, several ribosomes migrate along the length of the messenger RNA. They translate it simultaneously so that each one produces a complete polypeptide chain.

The ER is a double membrane system partitioning the cytoplasm. It is linked to the cytoplasmic membrane and nuclear membrane. It is, in a way, an extension of the latter. Although less developed in yeasts than in exocrine gland cells of higher eukaryotes, the ER has the same function. It ensures the routing of the proteins synthesized by the attached ribosomes. As a matter of fact, ribosomes can be either free in the cytosol or bound to the ER. The proteins synthesized by free ribosomes remain in the cytosol, as do the enzymes involved in glycolysis. Those produced in the ribosomes bound to the ER have three possible destinations: the vacuole, the plasma membrane, and the external environment (secretion). The presence of a signal sequence (a particular chain of amino acids) at the N‐terminal extremity of the newly formed protein determines the association of the initially free ribosomes in the cytosol with the ER. The synthesized protein crosses the ER membrane by an active transport process called translocation. This process requires the hydrolysis of an ATP molecule. Having reached the inner space of the ER, the proteins undergo certain modifications including the necessary excising of the signal peptide by signal peptidase. In many cases, they also undergo a glycosylation. The yeast glycoproteins, in particular the structural or enzymatic cell wall mannoproteins, contain carbohydrate side chains ( Section 1.2.2). Some of these are linked to asparagine by N ‐glycosidic bonds. This oligosaccharidic linkage is constructed in the interior of the ER by the sequential addition of activated sugars (in the form of UDP derivatives) to a hydrophobic lipid transporter called dolichol phosphate. The entire unit is transferred in one piece to an asparagine residue of the polypeptide chain. Thus, the dolichol phosphate is regenerated.

The Golgi apparatus consists of a stack of membrane sacs and associated vesicles. It is an extension of the ER. Transfer vesicles transport the proteins issued from the ER to the sacs of the Golgi apparatus. The Golgi apparatus has a dual function. It is responsible for completing the glycosylation of proteins and then it sorts them so as to direct them via specialized vesicles either into the vacuole or into the plasma membrane. An N‐terminal peptide (propeptide) sequence determines the routing of proteins toward the vacuole. This sequence is revealed in the precursors of two cytoplasm‐to‐vacuole targeting enzymes in the yeast: carboxypeptidase Y and proteinase A. However, the vesicles that transport the proteins of the plasma membrane or the secretion granules, like those that transport periplasmic invertase, are still the default destinations.

The vacuole is a spherical organelle, 0.3–3 μm in diameter, surrounded by a single membrane. Depending on the stage of the cellular cycle, yeasts have one or several vacuoles. Before budding, a large vacuole splits into small vesicles. Some penetrate into the bud, while others gather at the opposite end of the cell and fuse to form one or two large vacuoles. The vacuolar membrane, or tonoplast, has the same general structure (fluid mosaic) as the plasma membrane, but it is more elastic and its chemical composition is somewhat different. It is less rich in sterols and contains less protein and glycoprotein but more phospholipids with a higher degree of unsaturation. The vacuole stores some of the cell hydrolases, in particular carboxypeptidase Y, proteases A and B, aminopeptidase I, X‐prolyl‐dipeptidylaminopeptidase, and alkaline phosphatase. In this respect, the yeast vacuole can be compared to an animal cell lysosome. Vacuolar proteases play an essential role in the turnover of cellular proteins. In addition, protease A is indispensable in the maturation of other vacuolar hydrolases. It excises a small peptide sequence and thus converts precursor forms (proenzymes) into active enzymes. The vacuolar proteases are also responsible for autolysis, after cell death, when aging white wine on its lees.

Vacuoles also have a second principal function: they store metabolites before their use. In fact, they contain a quarter of the pool of amino acids of the cell, including a lot of arginine as well as S ‐adenosyl methionine. In this organelle, there is also potassium, adenine, isoguanine, uric acid, and polyphosphate crystals. These are involved in the fixation of basic amino acids. Specific permeases ensure the transport of these metabolites across the vacuolar membrane. An ATPase linked to the tonoplast furnishes the necessary energy for the movement of stored compounds against the concentration gradient. It is different from the plasma membrane ATPase, but also produces a proton efflux.

The ER, Golgi apparatus, and vacuoles must therefore be considered as different components of an internal system of membranes, called the vacuome, participating in the flux of glycoproteins to be excreted or stored.