The role of GRASP55 in Golgi fragmentation and entry of cells into mitosis.
GRASP55 is a Golgi-associated protein, but its function at the Golgi remains unclear. Addition of full-length GRASP55, GRASP55-specific peptides, or an anti-GRASP55 antibody inhibited Golgi fragmentation by mitotic extracts in vitro, and entry of cells into mitosis. Phospho-peptide mapping of full-length GRASP55 revealed that threonine 225 and 249 were mitotically phosphorylated. Wild-type peptides containing T225 and T249 inhibited Golgi fragmentation and entry of cells into mitosis. Mutant peptides containing T225E and T249E, in contrast, did not affect Golgi fragmentation and entry into mitosis. These findings reveal a role of GRASP55 in events leading to Golgi fragmentation and the subsequent entry of cell into mitosis. Surprisingly, however, under our experimental conditions, >85% knockdown of GRASP55 did not affect the overall organization of Golgi organization in terms of cisternal stacking and lateral connections between stacks. Based on our findings we suggest that phosphorylation of GRASP55 at T225/T249 releases a bound component, which is phosphorylated and necessary for Golgi fragmentation. Thus, GRASP55 has no role in the organization of Golgi membranes per se, but it controls their fragmentation by regulating the release of a partner, which requires a G2-specific phosphorylation at T225/T249.
Duran
Juan Manuel
JM
Cell and Development Program, Centro de Regulacion Genomica, 08003 Barcelona, Spain.
Kinseth
Matt
M
Bossard
Carine
C
Rose
David W
DW
Polishchuk
Roman
R
Wu
Christine C
CC
Yates
John
J
Zimmerman
Timo
T
Malhotra
Vivek
V
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D002457:Cell Extracts; D002460:Cell Line; D003001:Cloning, Molecular; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D000077614:Golgi Matrix Proteins; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008938:Mitosis; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats
D002457:Cell Extracts; D000077614:Golgi Matrix Proteins; C485684:Gorasp2 protein, rat; D008565:Membrane Proteins; D010750:Phosphoproteins; D034741:RNA, Small Interfering