title: Phosphorylation of Chs2p regulates interaction with COPII.
abstract: Trafficking of the chitin synthase Chs2p from the endoplasmic reticulum (ER) to the bud-neck in late mitosis is tightly regulated by the cell cycle via phosphorylation of serine residues in the N-terminus of the protein. Here, we describe the effects of Chs2p phosphorylation on the interaction with coat protein complex II (COPII). Identification of a cdc5(ts) mutant, which fails to transport Chs2p-3xGFP to the bud-neck and instead accumulates the protein in intracellular puncta, led us to discover that Chs2p-3xGFP accumulates at ER exit sites in metaphase-arrested wild-type cells. Using an in vitro ER vesicle formation assay we showed that phosphorylation of Chs2p by the cyclin-dependent kinase CDK1 prevents packaging into COPII vesicles, whereas dephosphorylation of Chs2p by the phosphatase Cdc14p stimulates selection into the vesicles. We found that the cytoplasmic N-terminal domain of Chs2p, which contains the CDK1 phosphorylation sites, interacts with the COPII component Sec24p in a yeast two-hybrid assay and that phosphomimetic substitutions of serines at the CDK1 consensus sites reduces the interaction. Our data suggest that dephosphorylation functions as a molecular switch for regulated ER exit of Chs2p.
authors.affiliation: Department of Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA.
authors.forename: Mia Kyed
authors.initials: MK
authors.lastname: Jakobsen
authors.forename: Zhiliang
authors.initials: Z
authors.lastname: Cheng
authors.forename: Sheung Kwan
authors.initials: SK
authors.lastname: Lam
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Roth-Johnson
authors.forename: Robyn M
authors.initials: RM
authors.lastname: Barfield
authors.forename: Randy
authors.initials: R
authors.lastname: Schekman
mesh_terms: D032701:Biomimetics; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D022181:COP-Coated Vesicles / Q000502:physiology*; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002687:Chitin Synthase / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005818:Genetic Engineering; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation / Q000235:genetics; D011485:Protein Binding; D021381:Protein Transport / Q000235:genetics; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins
chemical_list: C074860:CDC14 protein, S cerevisiae; D018797:Cell Cycle Proteins; D008565:Membrane Proteins; C118289:SEC24 protein, S cerevisiae; C106113:SEC31 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D002687:Chitin Synthase; C582195:Chs2 protein, S cerevisiae; D016203:CDC2 Protein Kinase; D017027:Protein Tyrosine Phosphatases
keywords: COPII; Cell cycle; Chs2p; ER; Protein phosphorylation; Secretory pathway