Yeasts have developed three different ways of attaching proteins to cell wall glucan. Some proteins are bound to beta-1,3-glucan non-covalently, while others are attached covalently, through GPI-anchor and beta-1,6-glucan, or directly to beta-1,3-glucan by alkali-labile ester linkage between the gamma-carboxyl groups of glutamic acid and the hydroxyl groups of glucoses (Pir proteins). In order to obtain further insight into the binding mechanism, a novel, simple binding assay for Pir-family proteins was developed. It has been shown that PIR, as well as SCW4 mutants, can bind externally added Ccw5p to their cell walls. A study of appropriate binding conditions revealed the requirement of the native conformation of Ccw5p. The presence of EDTA blocked the binding of Ccw5p, indicating the cation dependence of the reaction. Both wild-type and mutant cells showed enhanced binding of the Ccw5p in 0.6 M KCl. After disruption of all Pir genes (CCW5, CCW6, CCW7 and CCW8), 67 kDa protein still remained in NaOH extract. SCW4 disruption in the ccw5ccw6ccw7ccw8 mutant resulted in disappearance of the 67 kDa band from the extract, indicating that Scw4p could also be covalently linked to the cell wall by a so-far unidentified alkali-labile linkage.