In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase.

Abstract

Receptor-mediated endocytosis and recycling are inhibited in mitotic mammalian cells, and previous studies have shown that inhibition of endocytic vesicle fusion in vitro occurs via cyclin B-cdc2 kinase. To test for the ability of cyclin A-cdc2 kinase to inhibit endocytic vesicle fusion, we employed recombinant cyclin A proteins. Addition of cyclin A to interphase extracts activated a histone kinase and markedly reduced the efficiency of endocytic vesicle fusion. By a number of criteria, inhibition of fusion was shown to be due to the action of cyclin A, via the mitosis-specific cdc2 kinase, and not an indirect effect through cyclin B. Two-stage incubations were used to demonstrate that at least one target of cyclin A-cdc2 kinase is a cytosolic component of the fusion apparatus. Reconstitution experiments showed that this component was also modified in mitotic cytosols and was unaffected by N-ethyl maleimide treatment.

Molecular mechanisms in the disassembly and reassembly of the mammalian Golgi apparatus during M‐phase

“…Inhibition of vesicle fusion is consistent with the observed general cessation of exocytic and endocytic transport during M-phase [10]. Work on endocytosis suggests that the inhibited step in the transport event is the fusion of vesicles with their target membrane [32,33]. In the case of the Golgi apparatus, inhibition of vesicle fusion would, of course, result in the accumulation of COP I vesicles in the vicinity of Golgi stacks.…”

Oncogenic activation of a human cyclin A2 targeted to the endoplasmic reticulum upon Hepatitis B virus genome insertion

“…Cyclin A2 associated H1 kinase activity has been reported in the endosomal as well as ER fractions of rat liver cells and is increased in vivo during liver regeneration (Castro et al, 1994;Verges et al, 1997). It has been suggested that the kinase activity associated with cytosolic cyclin A2 might be implicated in the inhibition of in vitro intra-Golgi transport (Stuart et al, 1993;Mackay et al, 1993) and in the inhibition of the in vitro fusion of endocytic vesicles (Woodman et al, 1993), in contrast with previous reports (Thomas et al, 1992). The actual implications of these ®ndings for cell cycle progression have however not been established.…”

Clathrin-mediated endocytosis is inhibited during mitosis

“…Further studies demonstrated that uptake of fluorescent dextran ceases during mitosis and also confirmed the timing of shutdown to be in early prophase (7). Many other studies went on to confirm and further develop the theory of mitotic shutdown of endocytosis, showing that it is temporary and resumes in telophase (8)(9)(10)(11)(12)(13)(14)(15)(16)(17). The reason for shutdown is unclear (18,19), but one recent idea is that "moonlighting" functions for endocytic proteins during mitosis may contribute to the inhibition (20,21).…”