Saccharomyces Cerevisiae Png1p In The Yeast In Vivo Properties Of,

Peptide:N-glycanase (PNGase) of eukaryotic cells, Png1p, is a kind of Glycanase which recognizes and deglycosylates non-native /V-linked glycoproteins .The weight and structure of various Png1p are different, but they all have highly conserved catalyze cores. The Png1p of 5. cerevisiae is simple. It is known that Png1p plays an important role in the Glycoproteins ER-associated Degradation, GERAD. But it is not clear that how Png1p recognizes the false fold glycoproteins, how it plays a part in the GERAD. And we don't know the influence of Png1p on the cell's living either.In order to answer questions above, two experiments have been done.First, the interaction between Png1p and other proteins in yeast is researched. It is reported that most of Png1p exists mainly in cytoplasm, and few locates on the ER membrane by Png1p-GFP fusion protein. The two kinds of Png1p may play different role in GERAD. There have been several hypotheses about the role of Png1p in GERAD. The research of interaction between Png1p and other proteins is the key to prove whether the hypotheses are true. With a new kind of Bacterial two-hybrid system which uses GFP-fragment reassembly as a report gene, interaction between Png1p and certain proteins is researched. Otherwise, a gene library is constructed to find interactions between Png1p and unknown proteins. Finally, the interaction between Png1p and Rad23 is proved.Second, the influence of gene disruption of Png1 in S. cerevisiae is studied. With a Cre-loxP gene knockout system, Png1 is replaced with KanMX by homologous recombination. Then, the KanMX between pairs of loxP sites is cutted by P1 bacteriophage cyclization recombination recombinase(Cre), and KanMX can be used as a screen marker again. And then, the growth of Png1-knockout S. cerevisiae is observed. The DNA repair function of Rad23 is tested. The result is that disruption of Png1 has no big influence on S. cerevisiae. So that, Png1p may not be necessary to yeast, and there may be other ways to deglycosylate glycoproteins.