Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes
numerous human pathologies. Biochemical evidence suggests that soluble misfolded
proteins are retrotranslocated out of the ER, via the endoplasmic reticulum associated
degradation (ERAD) pathway, for proteosome-mediated cytoplasmic degradation.
Excess, misfolded- or insoluble proteins, are suggested to cause induction of “overflow”
degradation pathways. For soluble proteins, overflow to vacuole-mediated destruction is
suggested to occur via two Golgi-to-vacuole (Gvt) routes, the alkaline phosphatase
(ALP), direct route, or, a carboxypeptidase Y- (CPY-), prevacuolar compartmentvacuole,
indirect route, though only the CPY route is thought to degrade soluble
proteins. Insoluble aggregate-containing structures are suggested to be degraded by
engulfment by membranes of unknown origin and trafficking to the vacuole for
destruction, via an autophagic pathway. To confirm biochemical evidence, wild-type
(BY4742), autophagosome- (W303/ATG14), CPY- and autophagy pathway-
(W303/VPS30), and proteosome (WCG/2) mutants of S. cerevisiae yeasts were
transformed with a high expression pYES plasmid and mutant (Z) human alpha-1-
proteinase inhibitor (A1PiZ), giving rise to the derivatives cells BY4742/Z,
W303/ATG14/Z, W303/VPS30/Z and WCG/2/Z, respectively. Electron microscopy
using gold labeling for A1PiZ, markers for the ER, the ERAD ER channel protein,
Sec61, or the chaperone, binding protein (BiP), ALP for the ALP pathway, and CPY for
the CPY pathway, was used. Overexpression of A1PiZ seems to result in targeting to
the vacuole via a prevacuolar, CPY-like compartment (PVC, 200-500 nm), though CPY
and A1PiZ appears not to colocalise, unconvincingly confirming collaborative
biochemical data. Large amounts of A1PiZ localise in the cytosol, possibly indicating a
largely proteasome-mediated degradation. ER-resident A1PiZ targeting to the vacuole
seems also to occur by the budding of the ER and peripheral plasma membrane or ER
membrane only. This occurs in all cells, but especially in ATG14 gene (ΔATG14)
mutants, possibly indicating autophagosome-mediated degradation independence, in the
latter mutants. The ATG14 mutation gave rise to crescent-shaped, initiating membranelike
(IM-like) structures of approximately Cvt vesicle-diameter, possibly indicating that
ΔATG14 blocks autophagosome- (500-1000 nm) and Cvt vesicle (100-200 nm) enclosure, after core IM formation. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/10839 |
| Date | January 2008 |
| Creators | Lamberti, Kelvin Robert. |
| Contributors | Elliott, Edith. |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
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