Abstract
BackgroundVirus infected killer strains of the baker’s yeast Saccharomyces cerevisiae secrete protein toxins such as K28, K1, K2 and Klus which are lethal to sensitive yeast strains of the same or related species. K28 is somewhat unique as it represents an α/β heterodimeric protein of the A/B toxin family which, after having bound to the surface of sensitive target cells, is taken up by receptor-mediated endocytosis and transported through the secretory pathway in a retrograde manner. While the current knowledge on yeast killer toxins is largely based on genetic screens for yeast mutants with altered toxin sensitivity, in vivo imaging of cell surface binding and intracellular toxin transport is still largely hampered by a lack of fluorescently labelled and biologically active killer toxin variants.ResultsIn this study, we succeeded for the first time in the heterologous K28 preprotoxin expression and production of fluorescent K28 variants in Pichia pastoris. Recombinant P. pastoris GS115 cells were shown to successfully process and secrete K28 variants fused to mCherry or mTFP by high cell density fermentation. The fluorescent K28 derivatives were obtained in high yield and possessed in vivo toxicity and specificity against sensitive yeast cells. In cell binding studies the resulting K28 variants caused strong fluorescence signals at the cell periphery due to toxin binding to primary K28 receptors within the yeast cell wall. Thereby, the β-subunit of K28 was confirmed to be the sole component required and sufficient for K28 cell wall binding.ConclusionSuccessful production of fluorescent killer toxin variants of S. cerevisiae by high cell density fermentation of recombinant, K28 expressing strains of P. pastoris now opens the possibility to study and monitor killer toxin cell surface binding, in particular in toxin resistant yeast mutants in which toxin resistance is caused by defects in toxin binding due to alterations in cell wall structure and composition. This novel approach might be easily transferable to other killer toxins from different yeast species and genera. Furthermore, the fluorescent toxin variants described here might likewise represent a powerful tool in future studies to visualize intracellular A/B toxin trafficking with the help of high resolution single molecule imaging techniques.
Highlights
Virus infected killer strains of the baker’s yeast Saccharomyces cerevisiae secrete protein toxins such as K28, K1, K2 and Klus which are lethal to sensitive yeast strains of the same or related species
Internalization of K28 by sensitive yeast cells is realized in a two step mechanism: while α-1,3-linked cell wall mannoproteins are used as primary K28 binding sites at the outer yeast cell surface, the secondary plasma membrane receptor of K28 has recently been identified as the HDEL-receptor Erd2p [10, 11] which ensures endocytotic toxin uptake and retrograde transport through the secretory pathway [9]
For in vivo expression of the chimeric K28 variant, the methylotropic yeast P. pastoris was used as host to ensure proper posttranslational toxin processing and folding in the eukaryotic secretory pathway, reflecting the natural situation of toxin secretion in K28 killer strains of S. cerevisiae [26]
Summary
Virus infected killer strains of the baker’s yeast Saccharomyces cerevisiae secrete protein toxins such as K28, K1, K2 and Klus which are lethal to sensitive yeast strains of the same or related species. Further proteolytic preprotoxin processing in the late Golgi catalysed by the activities of Kex2p and Kex1p results in the formation and final secretion of a disulphide-bonded α/β heterodimeric protein toxin whose β-subunit carries a carboxyterminal ER retention motif (HDEL) which is essential for host cell intoxication and intracellular toxin transport [5, 8, 9]. Internalization of K28 by sensitive yeast cells is realized in a two step mechanism: while α-1,3-linked cell wall mannoproteins are used as primary K28 binding sites at the outer yeast cell surface, the secondary plasma membrane receptor of K28 has recently been identified as the HDEL-receptor Erd2p [10, 11] which ensures endocytotic toxin uptake and retrograde transport through the secretory pathway [9]. Since the dimeric α/β structure is characteristic for A/B toxin family members including clinically relevant representatives like cholera, anthrax and Shiga toxin, K28 represents an attractive model to study A/B toxin trafficking in yeast [16, 17]
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