Isolation and biochemical characterization of cell wall tight protein complex involved in self-flocculation of Kluyveromyces bulgaricus

Flocculation of yeasts is a cell-cell aggregation phenomenon which is drive n by interactions between cell wall lectins and cell wall heteropolysacchar ides. In Sabouraud medium, Kluyveromyces bulgaricus was highly flocculent. Incubation of flocculent K. bulgaricus cells with EDTA or Hecameg (R) led t o extracts showing hemagglutinating and flocculating properties. Purificati on of the extracts by native PAGE gave two bands which allowed flocculation of deflocculated K. bulgaricus. Both bands with specific reflocculating ac tivity were composed of five subunits, of which only three possessed weak r eflocculating activity upon deflocculated yeast. The mixture of these three proteins allow the recovery of initial specific reflocculating activity of the complex. These three proteins, denoted p28, p36 and p48, presented, in their first 15 amino acids, homologies with glycolysis enzymes, i.e., 3-ph osphoglycerate mutase, glyceraldehyde-3-phosphate dehydrogenase and enolase , respectively. However, no such enzymatic activity could be detected in th e crude extract issued from treatment with EDTA and Hecameg (R) of floccule nt yeast cells. When yeasts had grown in glucose poor medium, flocculation was drastically affected. The EDTA and Hecameg (R) crude extracts showed we ak reflocculating activity. After PAGE, the protein complexes did not appea r in the EDTA extract, but they did appear in the Hecameg (R) crude extract . These results suggest that: (i) self-flocculation of K. bulgaricus depend s on the expression of different floc-forming protein complex, (ii) these p roteins are galactose specific lectins showing homologies in their primary structure with glycolysis enzymes.