In this study, we conducted a survey of native secreted K. lactis proteins that are capable of binding to chitin or that have chitinolytic activity. We discovered an abundantly expressed and previously undescribed extracellular protein (KlCts1p) that cross-reacted with an anti-ChBD polyclonal antibody, bound chitin with high affinity, and exhibited a chitinase activity distinct from that of the secreted killer toxin. We cloned and sequenced KlCTS1 and showed that it encodes a family 18 chitinase with a type 2 chitin-binding domain. We demonstrated that the ChBD derived from KlCts1p functions independent of the catalytic domain and readily dissociates from chitin in an alkaline environment. Finally, we showed that K. lactis cells require KlCts1p for proper cytokinesis, suggesting that cells use this protein to degrade septal chitin.
The small size (~5 to 7 kDa), the substrate binding specificity, and the high avidity of ChBDs for chitin have led to utilization of these domains as affinity tags for immobilization of proteins to chitin surfaces. For example, the B. circulans
chitinase A1 type 3 ChBD has been used to immobilize fusion proteins on chitin beads (9
) and on chitin-coated microtiter dishes (3
). However, a drawback of using the B. circulans
ChBD as an affinity tag is that its binding to chitin is generally irreversible, which limits its utility for many applications, such as protein purification. In this study, we showed that the ChBD derived from KlCts1p can (i) bind to chitin in the absence of the catalytic domain, (ii) function as an affinity tag on a heterologously expressed protein in K. lactis
, and (iii) dissociate from chitin in 20 mM NaOH, whereas BcChBD cannot do this. The latter observation likely reflects intrinsic structural differences between type 2 and type 3 ChBDs. For example, type 3 ChBDs (including BcChBD) contain conserved hydrophobic and aromatic amino acids that likely mediate their interaction with chitin (14
), whereas type 2 ChBDs (including KlChBD) contain six conserved cysteine residues that mediate the tertiary structure, most likely through the formation of disulfide bridges (2
). This observation also suggests that KlChBD could be used as an affinity tag for reversible immobilization or purification of alkaliphilic or alkali-tolerant proteins. Such proteins are widely used in the laundry detergent industry, where enzymes that function at a high pH (e.g., cellulases and proteases) are utilized to degrade common stains (15
). Alternatively, it is possible to generate KlChBD mutants that dissociate from chitin at neutral pH. The plausibility of using mutagenesis to alter the chitin-binding characteristics of a ChBD is supported by a recent study in which a mutant type 3 ChBD that requires 2 M NaCl to bind chitin was created (9
The ideal yeast strain background for secretion of recombinant ChBD-tagged proteins would (i) produce no abundant chitin-binding proteins or chitinolytic activity that would copurify during downstream applications, (ii) be capable of achieving a high cell density in culture, and (iii) efficiently secrete recombinant proteins. Our data indicate that despite a mild growth phenotype, K. lactis Δcts1 GG799 cells are capable of achieving the same cell density as wild-type cells in culture and produce no proteins with detectable chitin-binding or chitinolytic activities. Additionally, we have recently found that Δcts1 cells retain the ability to abundantly secrete recombinant maltose-binding protein (B. Taron, P. Colussi, and C. Taron, unpublished data). Thus, the K. lactis GG799 Δcts1 strain is well suited as a host background for production of recombinant ChBD-tagged proteins.
Our data suggest that KlCts1p is required for proper cytokinesis of K. lactis
cells. Disruption of CTS1
in a haploid K. lactis
strain produced viable cells that had a separation defect. During growth of this strain, cells remained joined together via their septa, which stained brightly for the presence of chitin. Thus, it is probable that KlCts1p is required for removal of septal chitin during cytokinesis. A similar phenotype has been described for S. cerevisiae
). Therefore, we showed that KlCTS1
could restore normal morphogenesis to S. cerevisiae
cells, indicating that KlCts1p and ScCts1p perform identical functions and that the two organisms likely have similar mechanisms for removing septum-localized chitin during cytokinesis.
Only a small fraction of total KlCts1p activity produced by K. lactis
cells may be required for cytokinesis. Exogenous addition of purified KlCts1p to the culture medium prior to growth of K. lactis
cells only partially alleviates the cell separation defect. Thus, exogenous KlCts1p may have a limited ability to access and cleave chitin in the septum. It is therefore possible that during secretion, some KlCts1p is retained in the cell wall, where it may act upon cell wall chitin during cell division. In support of this notion, we found that 99% of KlCts1p activity localizes to the culture medium, whereas ~1% remains associated with a cell wall fraction of lysed cells (data not shown). Thus, it is likely that efficient cytokinesis requires only limited hydrolysis of cell wall chitin by KlCts1p. However, the abundance of medium-localized KlCts1p may reflect a second purpose for the protein. It is possible that secreted KlCts1p acts as an antimicrobial agent by digesting exposed chitin on competing fungi. Such a function was suggested by experiments in which transgenic tobacco plants expressing the S. cerevisiae
Cts1 chitinase could inhibit spore germination and hyphal growth of the fungal pathogen Botrytis cinerea