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Lysosomes are membrane bound organelles filled with hydrolytic
enzymes that the cell uses to digest various macromolecules. These
macromolecules include proteins, polysaccharides, fats, and nucleic
acids. Because these enzymes function optimally under acidic
conditions, the lysosomal environment must be of a lower pH than the
rest of the cell. This is accomplished through the pumping of hydrogen
ions from the cytosol into the lumen of the lysosome.
The lysosomal associated membrane protein (LAMP-1) and Cathepsin D
are both proteins found in the lysosome. While both proteins must
travel to the lysosome after synthesis, the pathways they follow to
their target destination are significantly different. Cathepsin D must
first be glycosylated in the lumen of the endoplasmic reticulum (ER)
in order to allow binding by mannose-6-phosphate to occur. The protein
then travels to the Golgi apparatus for the consequent steps of
glycosylation and must be bound by mannose-6-phosphate in order to
attach itself to the mannose-6-phosphate receptor. In contrast, the
LAMP-1 protein, although a heavily glycosylated protein, does not
follow the mannose-6-phosphate protein targeting path.
Exposing cells to tunicamycin disrupts glycosylation of
newly-synthesized proteins. This is due to the blockage of the
transference of the 14 residue core oligosaccharide (containing two N-acetylglucosamine,
nine mannose, and three glucose residues) from a dolichol phosphate
donor molecule to certain asparagine (Asn) residues on the proteins.
In the following investigation, cells treated with various
concentrations of tunicamycin at differing incubation times are used
to investigate the effects of glycosylation of newly-synthesized
proteins on protein transportation.
If glycosylation does not have any effect upon LAMP-1, then LAMP-1
should reach its target organelle. Although LAMP-1 does not require
glycosylation in order to be transported into the lysosome,
tunicamycin treatment could alter the protein's tertiary structure,
resulting in improper folding. If this occurs, LAMP-1 may be
depolarized in either the ER or the Golgi apparatus. This should cause
an accumulation of the protein in the ER. Lack of glycosylation and/or
misfolding may also lead to protein degradation, in which case the
protein would be destroyed and/or secreted. A loss of glycosylation
should signify rapid degradation and eventual secretion of Cathepsin
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