A caspase cleavage fragment of p115 induces fragmentation of the Golgi apparatus and apoptosis

Abstract

In mammalian cells the Golgi apparatus consists of a series of flattened cisternae localized to the perinuclear region of the cell. The Golgi undergoes extensive and reversible fragmentation into vesicles and tubules during mitosis; these reassemble into cisternae at cytokinesis. A protein complex that mediates post-mitotic Golgi re-assembly has been characterized by several laboratories and consists of at least four core, high molecular weight proteins: p115, GM130, GRASP65 and giantin. p115 has been demonstrated to play a key role in vesicle tethering and is required for maintaining the structural organization of the Golgi apparatus. Phosphorylation of GM130 disrupts its interaction with p115 and prevents vesicle tethering to the Golgi. This is thought to be the key event in effecting mitotic Golgi fragmentation.;In contrast to mitosis, the Golgi undergoes irreversible fragmentation during programmed cell death (apoptosis). A main goal of this thesis research was to determine if Golgi fragmentation during apoptosis and mitosis occurred via a similar mechanism. The data suggest that this is not the case. GM130 was not phosphorylated and caspases were found to cleave both p115 and GM130 during apoptosis. Unlike protein phosphorylation during mitosis, the irreversible nature of protein cleavage during apoptosis results in the permanent inhibition of vesicle reassembly into Golgi cisternae. Compared to control cells expressing wildtype p115, those expressing a caspase cleavage-resistant form of p115 delayed Golgi fragmentation during apoptosis. Expression of cDNAs encoding full-length or an N-terminal caspase cleavage fragment of p115 had no effect on Golgi morphology. In contrast, expression of the 30 kDa C-terminal caspase-cleavage product of p115 induced Golgi fragmentation. Furthermore, this fragment translocated to the nucleus and its expression was sufficient to induce apoptosis. Deletion analysis showed that expression of a core fragment comprising 102 amino acids of the 30 kDa polypeptide was able to translocate to the nucleus and induce apoptosis. Most significantly, in vivo expression of the C-terminal fragment in the presence of caspase inhibitors or upon co-expression with a cleavage-resistant mutant of p115 showed that p115 degradation plays a key role in amplifying the apoptotic response independently of Golgi fragmentation.;In addition to p115, other factors implicated in maintaining Golgi structure were also cleaved by caspases during apoptosis. Among them is the beta isoform of the phosphatidylinositol 4-phosphate 5-kinase (mPIP5KIbeta). Like p115, expression of the C-terminal caspase cleavage fragment of mPIP5KIbeta also resulted in its translocation into the nucleus and the induction of Golgi fragmentation and apoptosis. Expression of the full-length or the N-terminal cleavage fragment of mPIP5KIbeta had no effect on Golgi structure or cell survival. In sum, our data suggest that cleavage of a single substrate to effect multiple events may be a common theme in apoptosis. This idea emphasizes the efficiency in which the apoptotic program causes the ultimate demise of the cell.