Yeast Cell Envelopes Biochemistry Biophysics and Ultrastructure

Volume II

Leo H Arnold

• Publisher: CRC Press
• Year: 2018
• ISBN:
  9781351094689
• Edition: 1
• Topic: Biological Sciences
• Subtopic: Microbiology
• Index: Biological Sciences

A comprehensive review of the yeast cell envelope has not appeared previously and therefore this book is timely. The title of this volume was chosen to reflect the three major areas of contribution to our current understanding of the cell envelope, but we have not attempted to group chapters into subdivisions. The approach was to describe phenomena, to review the literature and to illuminate outstanding problems. It was also attempted to generate working hypotheses which may stimulate further studies. The some of these ideas be of germinal value is of more concern to us than that all of the hypotheses should stand the test of further experimentation.

Chapter 1

ENZYMES

W. N. Arnold

TABLE OF CONTENTS

I.	Background
II.	Projected Behavior of Cell Envelope Enzymes
III.	Enzymes of the Periplasmic Space
	A. Detection
	1. Kinetic Restraints
	2. Other Causes of Quantitative Error or Nondetection
	B. β-Fructofuranosidase
	1. Reaction
	2. Assay
	3. Location
	4. Chemical Structure and Properties
	C. Acid Phosphatase
	1. Reaction
	2. Assay
	3. Location
	4. Chemical Structure and Properties
	D. Other Enzymes of the Periplasmic Space
IV.	Enzymes of the Cell Wall
	A. Criteria and Detection
	B. Wall Preparations
	C. β-Glucanases
	D. Other Cell Wall Enzymes
V.	Enzymes of the Plasma Membrane
	A. Criteria and Detection
	B. Membrane Preparations
	C. Chitin Synthetase
	D. Mg^2+-Adenosine Triphosphatase
	E. Phospholipase
	F. Glucan Synthetase
	G. Other Possibilities
VI.	Enzymes Yet to Be Differentiated within Cell Envelopes
VII.	Enzymes Secreted into the Medium
VIII.	Biosynthesis of Cell Envelope Enzymes
	A. Repression, Derepression, and Induction
	B. Translocation
IX.	Concluding Remarks
Acknowledgment
References

I. BACKGROUND

The yeast cell envelope contains an important complement of enzymes. Self-evident as that statement is now, it was rather the mechanical role of the cell wall that dominated earlier biological considerations. Consequently the envelope was looked upon as a relatively inert complex of integuments rather than an “outer metabolic region of the cell.”¹

Studies on the enzymology of the yeast cell envelope have their beginning in turn of the century work on isolation and partial purification of β-fructofuranosidase. By 1921 Willstätter’s laboratory² had incidentally documented the peripheral location of β-fructofuranosidase, and other enzymes were subsequently associated with the cell envelope. These enzymes fall into two classes: first, those which cleave substrates that do not permeate the plasma membrane, and second, those enzymes involved with the turnover of envelope polymers during growth. The first group includes nutritionally important enzymes, and some members are well documented; while the second group includes both synthetic and degradative members that have been sparingly studied and, indeed, their existence in some cases is only inferred from experiments with inhibitors.

The methodology for studying enzymes of the cell envelope is generally common to that used with cell-free extracts. However, certain operational advantages accrue because of facile addition and later substraction of substrates or effectors. This is achieved by washing cells (or derivative structures) on the centrifuge or on filters. Conversely, macromolecular probes (enzymes for coupled-assay, labeled antibodies, etc.) are debarred from the periplasmic space by dint of the impermeability of the cell wall, and this may present certain operational disadvantages and problems in interpretation.

This review follows a division according to apparent location of enzymes within the cell envelope. Operational definitions for the component regions of the envelope have been given in Volume I, Chapter 1. It will become evident that the depth of characterization among the individual enzymes varies greatly.

II. PROJECTED BEHAVIOR OF CELL ENVELOPE ENZYMES

Certain criteria can be assembled as hallmarks of this group of enzymes. Other tests help to distinguish fine locations within the envelope, and meeting all or some of these criteria will more or less strengthen the assignment of a particular enzyme to a specific locale. The practical results which speak to these parameters are subject to interpretations that are summarized in succeeding sections. Table 1 is a summary of this type of analysis for the cell wall, periplasmic space, and plasma membrane. Capsulated yeasts may have some enzymes associated with the slime layer, but there is no information to this effect and in this chapter we can only mention certain salient points about the slime layer in passing.

Table 1
PROJECTED BEHAVIOR OF CELL ENVELOPE ENZYMES

Criterion	Cell wall	Periplasmic space	Plasma membrane
1. Accessibility to substrate without need of prior perturbation of membranes	+	+	+
2. Modification by reagents excluded by plasma membrane	+	+	+
3. Identity of pH profiles of cell and cell-free extract	+	+	+
4. Release of nonsedimentable activity by protoplasting	+^a	+	—
5. Release of nonsedimentable activity by cracking.	“	+
6. Secretion by protoplasts that are capable of de novo enzyme synthesis	+	+
7. Cytochemical localization	Yes	Yes	Yes
8. Artificial cross-linking to plasma membrane	-	+	N.A.

Note: (+) = Enzyme in this locale should meet the stated criterion; (–) = should not meet the criterion; and N.A. = not applicable.

^a May be (–) if enzyme is associated with bud scar residues. See Volume I, Chapter 5, Figures 6 and 7.

Criteria 1,2, and 3 arise from the fact that the cell wall is freely permeated by most low molecular weight substrates, hydrogen ions, and several potential inhibitors or inactivators, that normally do not cross the plasma membrane. (This subject is discussed further in Volume I, Chapter 3.) As well as demonstrating that a candidate enzyme is directly assayable in the intact cell, and that it feels the bathing medium, it becomes equally important to demonstrate that the same conditions do not support assay or modification of cytoplasmic enzymes. If the activity per cell is increased by pretreatments that perturb membranes, then an additional location may be indicated. However, such an increase may be deceptive because the perturbant may also function by inhibiting a secondary event (metabolic or transport) that would otherwise deplete the assayable product in question. This is discussed in more detail under kinetic restraints.

Useful inactivators under criterion 2 include H⁺ and heavy metals. The proper functioning of the plasma membrane (see also Volume I, Chapter 4) enables the cytoplasm to be maintained at a fairly constant pH in spite of external changes. Likewise, salts are excluded, especially if the bathing medium is free of metabolizable substrates. However, at extremes of pH the plasma membrane is perturbed and the differential between internal and external pH is abolished. That the integrity of the plasma membrane has not been compromised needs to be demonstrated by the proper controls if criteria 1 to 3 are to be interpreted meaningfully.

Criterion 4 follows from the fact that dissolution of the cell wall by exogenous glucanases will remove the network of the wall, and those enzymes associated with the periplasmic space and the cell wall will now freely diffuse into the bathing medium. Accordingly, the released enzyme will resist sedimentation by high-speed centrifugation. Important controls under this heading include documentation of incidental enzymes in the protoplasting mixture (e.g., snail digestive juice is endowed with many additional enzymes as well as the vital β-glucanases needed for yeast cell wall dissolution) and, in the case of a negative finding, checking the candidate enzyme for susceptibility of inactivation during the protoplasting treatment. Further discussion on protoplast formation is given in Volume II, Chapter 5, but it is worth mentioning here that osmotic vulnerability alone is not sufficient evidence for protoplast formation. That protoplasts devoid of cell wall remnants have indeed been produced needs to be confirmed by electron microscopy.

Release of the enzyme in question during protoplasting does not distinguish between the periplasmic space and the cell wall because the latter is dissolved by the treatment. Criterion 5 provides distinctive evidence in this context because mechanical disruption (See Volume I, Chapter 3 for specific methods) cracks the cell wall in a limited number of locations and large pieces of cell wall are discernible under the light microscope. If the candidate enzyme is associated with the cell wall it will be sedimented by centrifugation, whereas if the enzyme is located in the periplasmic space then but one crack in the wall will be a sufficient opening through which the enzyme can diffuse into the medium. In the latter case th...

Table of contents

• Cover
• Title Page
• Copyright Page
• Table of Contents
• Chapter 1: Enzymes
• Chapter 2: Biosynthetic Mechanisms for Cell Envelope Polysaccharides
• Chapter 3: Molecular Biology of Budding
• Chapter 4: Fission
• Chapter 5: Protoplasts
• Chapter 6: Morphogenesis in Protoplasts
• Chapter 7: Autolysis
• Chapter 8: Vegetative Ultrastructure
• Subject Index
• Taxonomic Index