Dataset Information

Sec16 function in ER export and autophagy is independent of its phosphorylation in Saccharomyces cerevisiae.

ABSTRACT: Coat protein complex II (COPII) protein assembles at the endoplasmic reticulum exit site (ERES) to form vesicle carrier for transport from the ER to the Golgi apparatus. Sec16 has a critical role in COPII assembly to form ERES. Sec16?565N mutant, which lacks the N-terminal 565 amino acids, is defective in ERES formation and ER export. Several phosphoproteomic studies have identified 108 phosphorylated Ser/Thr/Tyr residues in Sec16 of Saccharomyces cerevisiae, of which 30 residues are located in the truncated part of Sec16?565N. The exact role of the phosphorylation in Sec16 function remains to be determined. Therefore, we analyzed nonphosphorylatable Sec16 mutants, in which all identified phosphorylation sites are substituted with Ala. These mutants show ERES and ER export comparable to those of wild-type Sec16, although the nonphosphorylatable mutant binds the COPII subunit Sec23 more efficiently than the wild-type protein. Because nutrient starvation-induced autophagy depends on Sec16, Sec16?565N impairs autophagy, whereas the nonphosphorylatable mutants do not affect autophagy. We conclude that Sec16 phosphorylation is not essential for its function.

SUBMITTER: Yorimitsu T

PROVIDER: S-EPMC7001475 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Sec16 function in ER export and autophagy is independent of its phosphorylation in Saccharomyces cerevisiae.

Yorimitsu Tomohiro T Sato Ken K

Molecular biology of the cell 20191218 3

Coat protein complex II (COPII) protein assembles at the endoplasmic reticulum exit site (ERES) to form vesicle carrier for transport from the ER to the Golgi apparatus. Sec16 has a critical role in COPII assembly to form ERES. Sec16∆565N mutant, which lacks the N-terminal 565 amino acids, is defective in ERES formation and ER export. Several phosphoproteomic studies have identified 108 phosphorylated Ser/Thr/Tyr residues in Sec16 of Saccharomyces cerevisiae, of which 30 residu ...[more]

PMID: 31851588

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Project description:BackgroundHuman BiP is traditionally regarded as a major endoplasmic reticulum (ER) chaperone performing a number of well-described functions in the ER. In recent years it was well established that this molecule can also be located in other cell organelles and compartments, on the cell surface or be secreted. Also novel functions were assigned to this protein. Importantly, BiP protein appears to be involved in cancer and rheumatoid arthritis progression, autoimmune inflammation and tissue damage, and thus could potentially be used for therapeutic purposes. In addition, a growing body of evidence indicates BiP as a new therapeutic target for the treatment of neurodegenerative diseases. Increasing importance of this protein and its involvement in critical human diseases demands new source of high quality native recombinant human BiP for further studies and potential application. Here we introduce yeast Saccharomyces cerevisiae as a host for the generation of human BiP protein.ResultsExpression of a full-length human BiP precursor in S. cerevisiae resulted in a high-level secretion of mature recombinant protein into the culture medium. The newly discovered ability of the yeast cells to recognize, correctly process the native signal sequence of human BiP and secrete this protein into the growth media allowed simple one-step purification of highly pure recombinant BiP protein with yields reaching 10 mg/L. Data presented in this study shows that secreted recombinant human BiP possesses native amino acid sequence and structural integrity, is biologically active and without yeast-derived modifications. Strikingly, ATPase activity of yeast-derived human BiP protein exceeded the activity of E. coli-derived recombinant human BiP by a 3-fold.ConclusionsS. cerevisiae is able to correctly process and secrete human BiP protein. Consequently, resulting recombinant BiP protein corresponds accurately to native analogue. The ability to produce large quantities of native recombinant human BiP in yeast expression system should accelerate the analysis and application of this important protein.

Dataset Information

Sec16 function in ER export and autophagy is independent of its phosphorylation in Saccharomyces cerevisiae.

Publications

Sec16 function in ER export and autophagy is independent of its phosphorylation in <i>Saccharomyces cerevisiae</i>.

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