Isolation and characterization of Saccharomyces cerevisiae mutants for vacuolar import and autophagocytosis

by Luminita Cornelia Andrei
Doctoral thesis
Date of Examination:2000-01-26
Date of issue:2001-11-01
Referee:Prof. Dr. Dr. h.c. Kurt von Figura
Referee:Prof. Dr. Gerhard Braus
Persistent Address: http://dx.doi.org/10.53846/goediss-651

 

 

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Abstract

English

The yeast vacuolar protein aminopeptidase 1 (Apelp) is synthesised as a cytosolic procursor that is transported to the vacuole via the cytoplasm to vacuole trnasport (Cvt) pathway. The cytosolic protein is enclosed in a double-membrane vesicle, which is transported to and fuses with the vacuole releasing a single-membrane autophagic body into the vacuolar lumen. This is degraded and the precursor sequence of aminopeptidase 1 is removed. The Cvt pathway involves proteins that are involved also in macroautophagy. The cytosolic precursor protein and the matured vauolar protein from homododecameric complexes. Only the matured homododecameric complex shows enzymatic activity. We developed a new genetic screen to isolate mutants in the biogenesis of the vacuolar aminopeptidase 1 based on the enzymatic activity. We developed a new genetic screen to isolate mutants in the biogenesis of the vacuolar aminopeptidase 1 based on the enzymatic activity. New mutants defective in the tranpor! t of aminopeptidase 1 to the vacuole have been isolated. Two of them have been characterised in detail. Those have defects in the dodecamer formation of the precursor form and accumulate it in pre-vacuolar vesicles. This suggests interdependence between the dodecamer formation and the formation of transport competent Cvt vesicles. The tentative identification was based on the screening of a yeast genomic library and sub-clonin of the particular genes. The effects of overexpression of some heat shock proteins, which are known to assist protein translocation processes, protein folding and assembly, indicated the participation of these factors in early events of the Cvt-pathway.
 
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