title: Expression of mouse cathepsin L cDNA in Saccharomyces cerevisiae: evidence that cathepsin L is sorted for targeting to yeast vacuole.
abstract: To investigate the intracellular transport mechanism of lysosomal cathepsin L in yeast cells, we attempted to produce mouse cathepsin L in Saccharomyces cerevisiae by placing the coding region under the control of the promoter of the yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Immunoblotting analysis by the use of an antibody specific for rat cathepsin L revealed that the yeast cells carrying the cathepsin L coding sequence produced 39- and 30-kDa products, which correspond to the rat procathepsin L and the single-chain form of mature cathepsin L, respectively. The precursor polypeptide showed sensitivity toward endoglycosidase H treatment. Cell fractionation experiments demonstrated that the processed form of 30-kDa cathepsin L was found to be colocalized to the yeast vacuole with the marker enzyme carboxypeptidase Y in a Ficoll step gradient. In the prepared vacuolar fraction, a considerable amount of cathepsin L was revealed to be cofractionated with the vacuolar membranes. Furthermore, the phase separation experiments with Triton X-114 provide the first evidence showing that the mature form of cathepsin L polypeptide is strongly associated with the vacuolar membranes. Therefore, the present results suggest that the mouse cathepsin L precursor polypeptide is initially synthesized as the proenzyme in the yeast cells and then correctly delivered to the vacuole. During the intracellular sorting pathway, the procathepsin L would undergo the post-translational proteolytic processing step to generate the mature enzyme. Based on these lines of evidence, we propose that cathepsin L is recognized by mechanisms similar to those for the intracellular sorting and processing of vacuolar proteins in the yeast cells.
authors.affiliation: Division of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Nishimura
authors.forename: K
authors.initials: K
authors.lastname: Kato
mesh_terms: D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D003546:Cysteine Endopeptidases; D004247:DNA / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D015151:Immunoblotting; D051379:Mice; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014617:Vacuoles / Q000201:enzymology*
chemical_list: D011994:Recombinant Proteins; D004247:DNA; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534278:Ctsl protein, mouse; C534280:Ctsl protein, rat