A role for the Golgi vesicle protein p115 in apoptosis and mitosis

Date

2007

Authors

Mukherjee, Shaeri

Journal Title

Journal ISSN

Volume Title

Publisher

ProQuest Dissertations & Theses

YU Faculty Profile

Abstract

The Golgi apparatus exists as a series of membrane stacks arranged in a pericentriolar lace-like reticulum. In mammalian cells it undergoes irreversible fragmentation during apoptosis. Previous work from this laboratory showed that the vesicle tethering protein p115 is cleaved during apoptosis. Caspase-8 or -3 cleavage of p115 generates a C-terminal fragment (CTF) of ∼205 residues which translocates to the cell nucleus. When expressed in otherwise healthy cells the p115 CTF accumulates in the nucleus, causes Golgi fragmentation and induces full-blown apoptosis.;The structure and orientation of the Golgi apparatus is regulated by the actin and tubulin cytoskeleton which have also been implicated in inducing apoptosis. When apoptosis was induced by Fas receptor activation, Golgi fragmentation was demonstrated to be an earlier event than previously recognized and it preceded breakdown of the actin and tubulin cytoskeleton. In this case, p115 cleavage preceded any significant morphological or biochemical changes to either the actin or microtubule cytoskeleton. Our data also showed that the nuclear localization of the CTF was not a consequence of its overexpression. In response to apoptotic stimuli, an endogenous polypeptide that cross-reacted with an antibody to the p115 C-terminus was present in cell nuclei. Together these results showed that CTF nuclear translocation was an early response during apoptosis.;To determine if nuclear translocation were required for the induction of apoptosis, a chimera comprising of a nuclear export signal fused to the CTF was expressed in cells. While high levels of the chimeric protein accumulated in the cytoplasm, few if any cells had an apoptotic phenotype. This observation in concert with data on the endogenous CTF, strongly suggested that nuclear translocation of the p115 CTF is necessary for the apoptotic response. To identify the region of the p115 CTF that induces apoptosis, a series of nested N- and C-terminal deletions were expressed in cells. A region of 26 amino acids (residues 859-884) that are predicted to form a perfect alpha-helical structure, were found to be essential for conferring the apoptotic phenotype. Consistent with this observation, a CTF deletion mutant lacking these residues translocated to the nucleus but did not induce apoptosis significantly. Together these data identified the key residues in p115 that mediate apoptosis. Use of bioinformatics revealed that the p115 CTF possesses three potential SUMOylation sites. Strikingly, co-expression of the CTF with SUMO and UBC9, the sumo-ligase, demonstrated that indeed the CTF molecule was SUMOylated in vivo. Most significantly, this result provided a mechanism whereby the CTF translocated into nuclei and suggests its mode of action could occur at the level of transcription.;Analysis of the p115 N-terminus uncovered an entirely novel role for this molecule and suggested that it functions in targeting the Golgi apparatus to the centrosomal region of the cell. Bioinformatics revealed that the N-terminal region possesses an armadillo-like domain that could promote interaction with the cytoskeleton. Consistent with this prediction, p115 was present on centrosomes and/or mitotic spindle poles early during mitosis and co-localized with gamma-tubulin, a centrosomal marker. In addition, both in vitro and in vivo pull down experiments showed that residues 1-636 of p115 bound to gamma-tubulin and deletion analysis showed that the armadillo-like domain (residues 60-97) by itself could bind gamma-tubulin. Finally, expression of various N-terminal truncated fragments of p115 demonstrated that those residues which bound to gamma-tubulin in vitro, also co-localized with it at the centrosome in vivo. Together, these results strongly suggest that the N-terminus of p115 plays a key role in tethering the Golgi apparatus to the centrosome during mitosis and suggests a model whereby both the N- and C-terminal domains of this protein participate in positioning the organelle correctly.

Description

Keywords

Cellular biology.

Citation

Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1396.;Advisors: Dennis Shields.