Retention of Chs2p in the ER requires N-terminal CDK1-phosphorylation sites.
In budding yeast, the secretory pathway is constitutively transporting cargoes such as invertase and alpha-factor throughout the cell division cycle. However, chitin synthase 2 (Chs2p), another cargo of the secretory pathway, is retained at the endoplasmic reticulum (ER) during mitosis when the mitotic kinase activity is high. Chs2p is exported from the ER to the mother-daughter neck only upon mitotic kinase destruction, indicating that the mitotic kinase activity is critical for the ER retention of Chs2p. However, a key question is whether the mitotic kinase acts directly upon Chs2p to prevent its ER export. We report here that mutation of Ser residues to Glu at 4 perfect CDK1-phosphorylation sites at the N-terminus of Chs2p leads to its retention in the ER when the mitotic kinase activity is absent. Conversely, Ser-to-Ala mutations result in the loss of Chs2p ER retention even when mitotic kinase activity is high. The mere overexpression of the non-destructible form of the mitotic cyclin in G(1) cells can confine the wild-type Chs2p but not the Ser-to-Ala mutant in the ER. Furthermore, overexpression of the Ser-to-Ala mutant kills cells. Time-lapsed imaging revealed that Chs2p is exported from the ER rapidly and synchronously to the Golgi upon metaphase release. Our data indicate that direct phosphorylation of Chs2p by the mitotic CDK1 helps restrain it in the ER during mitosis to prevent its rapid export in an untimely manner until after sister chromatid occurs and mitotic exit executed.
Teh
Ee Mei
EM
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
Chai
Chuan Chung
CC
Yeong
Foong May
FM
D001692:Biological Transport; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002687:Chitin Synthase / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins; D008938:Mitosis; D010766:Phosphorylation; D004718:Saccharomycetales
D005656:Fungal Proteins; D002687:Chitin Synthase; C400821:chitin synthase 2; D016203:CDC2 Protein Kinase