Kinetic studies of killer toxin K1 binding to yeast cells indicate two receptor populations

Kurzweilová, H.; Sigler, Karel

doi:10.1007/bf00314477

Cited by 24 publications

(19 citation statements)

References 16 publications

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“…Under the similar conditions, K1 demonstrated comparable dynamics of binding. In our hands, the binding was somewhat slower than previously shown for K1 (28), when the half-time of adsorption was less than 1 min, and the saturation was completed within 5 min. These variations could be attributed to the different test strains and approaches used.…”

Section: Discussioncontrasting

confidence: 51%

Yeast β-1,6-Glucan Is a Primary Target for the Saccharomyces cerevisiae K2 Toxin

et al. 2015

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b Certain Saccharomyces cerevisiae strains secrete different killer proteins of double-stranded-RNA origin. These proteins confer a growth advantage to their host by increasing its survival. K2 toxin affects the target cell by binding to the cell surface, disrupting the plasma membrane integrity, and inducing ion leakage. In this study, we determined that K2 toxin saturates the yeast cell surface receptors in 10 min. The apparent amount of K2 toxin, bound to a single cell of wild type yeast under saturating conditions, was estimated to be 435 to 460 molecules. It was found that an increased level of ␤-1,6-glucan directly correlates with the number of toxin molecules bound, thereby impacting the morphology and determining the fate of the yeast cell. We observed that the binding of K2 toxin to the yeast surface receptors proceeds in a similar manner as in case of the related K1 killer protein. It was demonstrated that the externally supplied pustulan, a poly-␤-1,6-glucan, but not the glucans bearing other linkage types (such as laminarin, chitin, and pullulan) efficiently inhibits the K2 toxin killing activity. In addition, the analysis of toxin binding to the intact cells and spheroplasts confirmed that majority of K2 protein molecules attach to the ␤-1,6-glucan, rather than the plasma membrane-localized receptors. Taken together, our results reveal that ␤-1,6-glucan is a primary target of K2 toxin and is important for the execution of its killing property.T he production of antimycotic killer toxins has been observed in several yeast genera and proved to be a widespread phenomenon (1, 2). Killer strains of Saccharomyces cerevisiae secrete protein toxins derived from a family of double-stranded RNAs (dsRNAs). The toxins have been grouped into four types (K1, K2, K28, and Klus) based on their killing profiles and lack of crossimmunity (3, 4). Such proteins are able to kill the nonkiller yeast, as well as yeast of other killer types, while the toxin-producing cells remain immune to their own or to the same type of killers (4, 5). K1 toxin disrupts the regulated ion flux across the plasma membrane, leading to the death of sensitive yeast strains (6, 7). The killing action of K1 toxin involves at least two steps. During the first step, the toxin binds to the cell wall, whereas the second step leads to the translocation and insertion of the toxin into the plasma membrane (6). Beta-1,6-glucan was originally proposed to be a cell wall receptor for K1 (8). Analysis of several kre mutants demonstrated that decrease of the cell wall ␤-1,6-glucan level leads to K1 resistance, thus confirming the involvement of this type of glucan in toxin binding (9). During the second step, K1 toxin interacts with plasma membrane receptors and disrupts the functional integrity of the plasma membrane either by inducing the formation of new ion channels (7) or through the activation of existing potassium channels (10). Products of TOK1 (protein, forming the potassium ion channel) and KRE1 (glycoprotein, involved in ␤-glucan assembly) have be...

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Section: Discussioncontrasting

confidence: 51%

Yeast β-1,6-Glucan Is a Primary Target for the Saccharomyces cerevisiae K2 Toxin

et al. 2015

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“…This scenario bears similarities with the proposed model of K1 binding to yeast cells. The experimental evidence indicates a two-step mechanism corresponding to two classes of binding sites for K1, a low-affinity and high-velocity binding to the cell wall receptor and a high-affinity and low-velocity binding component ref lecting the energydependent binding to a secondary plasma membrane receptor and subsequent formation of an ion channel (23,24).…”

Section: Discussionmentioning

confidence: 99%

Stress proteins on the yeast cell surface determine resistance to osmotin, a plant antifungal protein

Yun

Zhao

Pardo

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

131

150

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Strains of the yeast Saccharomyces cerevisiae differ in their sensitivities to tobacco osmotin, an antifungal protein of the PR-5 family. However, cells sensitive to tobacco osmotin showed resistance to osmotin-like proteins purified from the plant Atriplex nummularia, indicating a strict specificity between the antifungal protein and its target cell. A member of a gene family encoding stress proteins induced by heat and nitrogen limitation, collectively called Pir proteins, was isolated among the genes that conveyed resistance to tobacco osmotin to a susceptible strain. We show that overexpression of Pir proteins increased resistance to osmotin, whereas simultaneous deletion of all PIR genes in a tolerant strain resulted in sensitivity. Pir proteins have been immunolocalized to the cell wall. The enzymatic digestion of the cell wall of sensitive and resistant cells rendered spheroplasts equally susceptible to the cytotoxic action of tobacco osmotin but not to other osmotin-like proteins, indicating that the cell membrane interacts specifically with osmotin and facilitates its action. Our results demonstrate that fungal cell wall proteins are determinants of resistance to antifungal PR-5 proteins.

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“…Because the binding of PMKT to cell wall receptors, (136)-␤-D-glucans, occurs in the first 2-3 min after toxin addition (6), the observed lag phase, which is necessary for changes in cell viability to be observed, probably involves several events (Fig. 4): adhesion to membrane receptors; formation of ion channels in the plasma membrane (10); metabolic and genetic changes, such as the dissipation of transmembrane electrochemical gradients (9) or the induction of stress response elements; and the formation of large membrane pores that allow the passage of large molecules either simultaneously to or sequentially with the formation of ion channels (10,11,52).…”

Section: Discussionmentioning

confidence: 99%

The Transcriptional Response of Saccharomyces cerevisiae to Pichia membranifaciens Killer Toxin

Santos¹,

Álvarez

Mauro³

et al. 2005

Journal of Biological Chemistry

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The transcriptional response of Saccharomyces cerevisiae to Pichia membranifaciens killer toxin (PMKT) was investigated. We explored the global gene expression responses of the yeast S. cerevisiae to PMKT using DNA microarrays, real time quantitative PCR, and Northern blot. We identified 146 genes whose expression was significantly altered in response to PMKT in a non-random functional distribution. The majority of induced genes, most of them related to the high osmolarity glycerol (HOG) pathway, were core environmental stress response genes, showing that the coordinated transcriptional response to PMKT is related to changes in ionic homeostasis. Hog1p was observed to be phosphorylated in response to PMKT implicating the HOG signaling pathway. Individually deleted mutants of both up-(99) and down-regulated genes (47) were studied for altered sensitivity; it was observed that the deletion of up-regulated genes generated hypersensitivity (82%) to PMKT. Deletion of down-regulated genes generated wild-type (36%), resistant (47%), and hypersensitive (17%) phenotypes. This is the first study that shows the existence of a transcriptional response to the poisoning effects of a killer toxin.Killer phenomena are widespread in yeasts. Killer toxins are proteins or glycoproteins that are lethal to sensitive strains of the same species and a different variety of other yeast genera. In this line, attention has focused mainly on the characterization of killer toxins from Saccharomyces cerevisiae (K1, K2, and K28) followed more recently by the investigation of yeasts such as Kluyveromyces lactis, Zygosaccharomyces bailii, Hanseniaspora uvarum, Pichia membranifaciens, Debaryomyces hansenii, Schwanniomyces occidentalis,.P. membranifaciens CYC 1106 is a strain originally isolated from fermenting olive brines with pronounced killer activity against a variety of yeast species (8) and fungi (9). Once the protein nature of the toxin produced was established, the secreted protein was purified from the supernatant of growing cultures of P. membranifaciens in the early stationary phase. Previous biochemical studies on the PMKT 2 mechanism of killing of sensitive yeast cells indicated that PMKT is an 18-kDa protein that interacts with the (1 3 6)-␤-D-glucans of the cell wall of sensitive yeasts (6, 10). Recently the killing mechanism of this killer toxin has been elucidated (10). Regardless of certain possible additional effects, the killer toxin of P. membranifaciens CYC 1106 acts by disrupting plasma membrane electrochemical gradients. The death of sensitive cells in the presence of killer toxin is characterized by a leakage of common physiological ions through non-regulated ion channels in the plasma membrane causing a discharge of cellular membrane potential and changes in ionic homeostasis in a way comparable to that of certain killer toxins (K1) (11). Non-selective channel formation has been suggested to be the cytotoxic mechanism of action of PMKT (10).Yeasts must cope with different adverse environmental conditions, including hea...

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Kinetic studies of killer toxin K1 binding to yeast cells indicate two receptor populations

Cited by 24 publications

References 16 publications

Yeast β-1,6-Glucan Is a Primary Target for the Saccharomyces cerevisiae K2 Toxin

Yeast β-1,6-Glucan Is a Primary Target for the Saccharomyces cerevisiae K2 Toxin

Stress proteins on the yeast cell surface determine resistance to osmotin, a plant antifungal protein

The Transcriptional Response of Saccharomyces cerevisiae to Pichia membranifaciens Killer Toxin

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