A systematic study of the cell wall composition of Kluyveromyces lactis

Abstract

In many ascomycetous yeasts, the cell wall is composed of two main types of macromolecules: (a) polysaccharides, with a high content of β-1,6-and β-1,3-linked glucan chains and minor amounts of chitin; and (b) cell wall proteins of different types. Synthesis and maintenance of these macromolecules respond to environmental changes, which are sensed by the cell wall integrity (CWI) signal transduction pathway. We here present a first systematic analysis of the cell wall composition of the milk yeast, Kluyveromyces lactis. Electron microscopic analyses revealed that exponentially growing cells of K. lactis supplied with glucose as a carbon source have a wall thickness of 64 nm, as compared to 105 nm when growing on 3% ethanol. Despite their increased wall thickness, ethanol-grown cells were more sensitive to the presence of zymolyase in the growth medium. Mass spectrometric analysis identified 22 covalently linked cell wall proteins, including 19 GPI-modified proteins and two Pir wall proteins. Importantly, the composition of the cell wall glycoproteome depended on carbon source and growth phase. Our results clearly illustrate the dynamic nature of the cell wall of K. lactis and provide a firm base for studying its regulation.

Up against the Wall: Is Yeast Cell Wall Integrity Ensured by Mechanosensing in Plasma Membrane Microdomains?

“…The latter value was calculated by multiplication of the mxe by a factor of 0.85. This factor was obtained as the ratio of the cell wall thickness determined by transmission electron microscopy (102 nm) (46) to the mxe of 120 nm of a sensor that just reaches the cell surface, among a set of Wsc1 rulers employed for live-cell measurements of cell wall thickness by AFM (22). Since the STR region of the sensors is assumed to have spring properties and the CRD may have a globular rather than a linear conformation, the factor thus corrects for the difference between a fully extended (unnatural) sensor and its three-dimensional structure in live yeast cells.…”

Cell Wall-Related Bionumbers and Bioestimates of Saccharomyces cerevisiae and Candida albicans

“…Considering that Pma1 is an integral membrane protein with 10 predicted transmembrane domains, this indicates that the plasma membrane is packed with Pma1 (84). Our data can further be used as a starting point for a more quantitative analysis of other yeasts such as Candida glabrata (62) and other Candida spp., the industrial yeast Kluyveromyces lactis (85), and the fission yeast Schizosaccharomyces pombe (86,87). Interestingly, the haploid yeast form of E. dermatitidis (exponential-phase cells growing in rich medium) has been studied in depth by serial sectioning followed by a quantitative three-dimensional structural analysis (36,37).…”

Atomic force microscopy – looking at mechanosensors on the cell surface

“…Central to this discovery was the use of genetic manipulations to address a conceptual problem: AFM is a surface-based technique, so how can it probe protein sensors (such as Wsc1) that are embedded within the cell wall? The yeast cell wall has a thickness of ,100 nm (Backhaus et al, 2010), whereas the maximal length of the extracellular part of the Wsc1 sensor is 86 nm, meaning it does not reach the outermost cell surface. Therefore, the mechanosensors were artificially elongated to a length of 153 nm by inserting a stiff serine/threonine-rich region from the Mid2 sensor.…”