You have been studying the lysosomal enzyme cathepsin D in mammalian cells. To look at the localization of the enzyme, you incorporate 32 P into the cells over a long period of time and collect fractions of the cell lysate corresponding to various organelles. Much to your surprise, you find cathepsin D was isolated in the ER fraction, and it contains 32 positive sugar side chains. However, when you pass this enzyme through an affinity chromatography column containing mannose-6-phosphate receptor conjugated to resin, the enzyme passes straight through. However, your cousin calls you up and tell you that she modified your cell line. She claims that she deleted your endogenous cathepsin D gene and transfected a plasmid containing a cathepsin D gene with a Lys-Asp-Glu-Leu (KDEL) sequence on the C-terminus. a) Why is the lysosomal enzyme found in the ER fraction rather than in the lysosomal fraction?b) Explain why the modified enzyme does not bind to the mannose-6-phosphate receptor.

Explanation:

1.

Cathepsin is involved in programmed cell death, it is a lysosomal enzyme and the reason it is found in the ER fraction instead of the lysosomal fraction is because of its c terminal KDEL sequence. this sequence ia a retrieval for protein and causes a backward movement of protein back to endoplasmic reticulum. that is why cathepsin seems to have localised in ER instead of the lysosomal fraction

2 It does not bind to mannose-6-phosohate receptor because this glycosylation process does not occur in the E.R but in Golgi bodies.  because of the KDEL sequence this glycosylation of cathepsin would not occur and cathepsin would go through affinity column without any form of  binding to receptor.If cathepsin can get to the Golgi then glycolysation with Mannose6phospate can occur.

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