IN-VIVO PROCESSING OF THE PRECURSOR OF THE MAJOR EXOGLUCANASE BY KEX2ENDOPROTEASE IN THE SACCHAROMYCES-CEREVISIAE SECRETORY PATHWAY

We have established the main post-translational modification of the ma jor exoglucanase of Saccharomyces cerevisiae as the enzyme progresses through the secretory pathway. The protein portion of the enzyme accum ulated by sec18 cells was about 2 kDa larger than that of the secreted enzyme. This precursor (form A) was stable when maintained in the end oplasmic reticulum but was processed to the mature form (form B) befor e the block imposed by the sec7 mutation. Sec7 cells, when incubated a l 37 degrees C, accumulated form B first, but upon prolonged incubatio n, form A was preferentially accumulated. When the supply of newly syn thesized exoglucanase was prevented by the addition of cycloheximide, the accumulated A was transformed into B in the presence of altered Se c7p that still prevented secretion. Conversion of A into B was prevent ed in the double mutant sec7 kex2-1, indicating that Kex2p is central to the in vivo processing. Consistent with this, a KEX2 deletion mutan t secreted form A exclusively. Conversion of A into B was also prevent ed in sec7 cells by the presence of dinitrophenol, a poison that deple tes ATP levels, indicating that processing is dependent upon intracell ular transport which involves ER --> Golgi and/or, at least, one intra -Golgi step(s). It follows that this transport step(s) is independent of functional Sec7p.