Targeting and covalent modification of cell wall and membrane proteins heterologously expressed in the diatom Cylindrotheca fusiformis (Bacillariophyceae)

Diatoms are unicellular organisms encased by silica-based cell walls that d isplay species-specific structures, Morphogenesis of diatom cell walls is b elieved to be controlled by a polysaccharide/protein-matrix that remains as sociated with mature cell walls, Recently, a family of calcium-binding glyc oproteins, the frustulins, has been identified as major diatom cell wall co mponent, Here we describe a transformation-based approach to investigate in tracellular targeting and function of frustulins, When epsilon-frustulin fr om the diatom Navicula pelliculosa is expressed in Cylindrotheca fusiformis , it is correctly targeted into the cell wall, Furthermore, the unique N-te rminus of epsilon-frustulin was properly modified, indicating that C. fusif ormis and N. pelliculosa contain homologous fi frustulin-processing proteas es, In a different transformation experiment, a modified version of the Chl orella kessleri hexose/H+ symporter bearing a bacterial biotinyl-acceptor d omain was expressed in C, fusiformis. The transporter became biotinylated i n vivo and was functionally incorporated into the plasma membrane, allowing C. fusiformis to take up C-14-glucose and C-14-glucosamine. Stage-specific radioactive labeling with this transformant revealed that secretion of fru stulins is strongly enhanced during cell wall development, The data present ed in this study demonstrate for the first time functional expression of a membrane protein and correct targeting of a cell wall protein heterologousl y expressed in a diatom cell.