The Molecular Mechanism Underlying The Role Of Pex14 Phosphorylation In Pexophagy In Pichia Pastoris

Peroxisomes are single membrane enclosed organelles of eukaryotic cells and are involved in wide range of metabolic functions including the ?-oxidation of fatty acids and the detoxification of hydrogen peroxide.The size,number,and function of peroxisomes can be influenced by the environment of the cell.Peroxisome homeostasis is achieved by the counterbalance between organelle biogenesis and degradation.The cellular processes of peroxisome division,proliferation,inheritance and protein transport are called peroxisome biogenesis.Pexophagy,the selective autophagy of peroxisomes,contributes to the regulation of the organelle abundance.This mode of pexophagy includes micropexophagy and macropexophagy.Pex14 is an important peroxisomal membrane protein,which is not only involved in the biogenesis of peroxisome,but also in the pexophagy.The Pex14 is a kind of phosphorylated protein,which exists in both phosphorylated and non-phosphorylated states in P.pastoris.However,it is not clear which kinase phosphorylates Pex14,the phosphorylated sites of Pex14 protein and how Pex14 phosphorylation affects peroxisome biogenesis and degradation.In this study,we used P.pastoris Pex14 phosphorylation mutant strains to explore whether Pex14 phosphorylation affects peroxisome biogenesis and autophagy by using several methods including growth curve assay,western blot,fluorescence microscopy,in vitro kinase assay,yeast two-hybrid,and Co-IP.The main work was as follows:1.Identification of key phosphorylation sites of Pex14 in P.pastorisIn the previous work of our laboratory,phosphorylation mass spectrometry sequencing of P.pastoris Pex14 was carried out.Under the condition of methanol induction,the amino acids at position T288,T289,S292,and S385 may be the phosphorylation sites of Pex14.We integrated phosphorylated and non-phosphorylated mutations such as Pex14(T288,289,S292A),Pex14(T288,289,S292D),Pex14(S385A),Pex14(S385D)into the ?pex14 genome to analyze whether phosphorylated mutants of Pex14 are involved in peroxisome biogenesis or pexophagy.We detected the molecular weight of Pex14 in the mutant strains and found that only the molecular weight of Pex14 S385 D was higher than that of the unmutated Pex14,so S385 was determined as the key phosphorylation site of Pex14.2.Effect of Pex14 phosphorylation on biogenesis and degradation of peroxisome2.1 Pex14 phosphorylation was not involved in the biogenesis of peroxisomeBy measuring the growth curve of each phosphorylated mutant strain,we found that the Pex14 phosphorylation mutation did not affect the normal growth of the strain both in methanol medium and oleic acid medium,indicating that Pex14 phosphorylation was not involved in the biogenesis of peroxisome.2.2 Pex14 phosphorylation was involved in the micropexophagyWe induced micropexophagy in each phosphorylated mutant strain.Peroxisome matrix protein Aox was used as a marker protein to detect whether peroxisome degradation was affected.At the biochemical level,we used western blot to detect the degradation of Aox and GFP-Cleavage to detect the degradation of GFP-Aox and other fusion proteins.The results showed that the degradation of Aox in ?pex14::Pex14(T288,289,S292D)-His and?pex14::Pex14(S385D)-His strain slowed down under the condition of micropexophagy,thus determining the effect of Pex14 phosphorylation modification on micropexophagy.However,the difference of GFP-Aox degradation into vacuoles between Pex14(S385A)and Pex14(S385D)strains was not as obvious as that at the biochemical level.2.3 Pex14 phosphorylation modification has little effect on macropexophagyWe induced macropexophagy in each phosphorylated mutant strain.We did not observe the significant effect of Pex14 S385 D on pexophagy by detecting the degradation of Aox in macropexophagy,the degradation of GFP-Aox by GFP-Cleavage experiment and the degradation of GFP-Pmp20 by fluorescence microscopy.We also induced non-selective autophagy in each mutant strain.By detecting the degradation of Aox,we also did not observe that Pex14 S385 D had a significant effect on non-selective autophagy.3.Searching for kinases which can phosphorylate Pex14 by in vitro kinase assayAccording to bioinformatics software,we predicted that Ck2 and Gsk3 may phosphorylate S385 of P.pastoris Pex14,so we constructed an expression vector to purify the recombinant protein Ck2,Gsk3,and Pex14.The in vitro kinase assay was used to detect whether Ck2 and Gsk3 phosphorylate Pex14 S385.But we could not purify soluble Gsk protein.At present,we have purified His-Ck?1,GST-Pex14 ?N,and GST-Pex14 ?NS385A proteins.Then we use the purified proteins for in vitro kinase assay.Autoradiography results show that GST-Pex14 ?N and GST-Pex14 ?NS385A have bands,indicating that Ck?1 can phosphorylate Pex14,but S385 may not be the only Ck2 phosphorylation site.In the follow-up experiment,we intend to construct a truncated Pex14 protein and determine whether S385 is the phosphorylation site of Ck?1 by in vitro kinase assay.4.Molecular mechanism of the effect of Pex14 phosphorylation on pexophagyFrom the results of western blot,we have determined that Pex14 phosphorylation can slow down micropexophagy.To explore whether Pex14 phosphorylation affects micropexophagy by affecting the interaction with Atg30,we also carried out a yeast two-hybrid experiment to detect the interaction between Atg30 and Pex14.The ATG30-p GADT7 and PEX14-p GBKT7 constructs were co-transformed into S.cerevisiae strain AH109.The results showed that Atg30 and Pex14 did not interact with each other in vitro.So we plan to construct the vector ZEOCIN-ATG30-GFP-p IB2,and transform it into ?pex14::Pex14(S385A)-His,?pex14::Pex14(S385D)-His and ?pex14::Pex14-His strains,respectively,and prepare to use GFP-Trap for Co-IP experiment,to further analyze whether Pex14 phosphorylation affects micropexophagy by affecting the interaction with Atg30.