Biochemical analysis of the factors controlling the process of membrane tubule formation from the Golgi complex

Weigert, Roberto (2000). Biochemical analysis of the factors controlling the process of membrane tubule formation from the Golgi complex. PhD thesis The Open University.



Membranous tubules are very abundant structures in living cells and form or are part of most intracellular organelles. The Golgi apparatus is mainly formed by tubules, which adopt different geometries and conformations. However, their physiological role has not yet been established and this is mainly due to the almost absolute lack of knowledge about the biochemical mechanisms regulating their formation, maintenance and disruption.

The aim of this thesis was to investigate in a systematic way these mechanisms. The first step has been to set up an in vitro morphological assay suitable for the visualisation of Golgi-associated tubules in isolated Golgi stacks. This assay was based on electron microscopy and specifically on negative staining of whole-mount preparations. It allowed both qualitative and quantitative analysis of the morphological changes of Golgiassociated tubules after in vitro incubations.

This assay was then used for screening several molecules or experimental conditions for their effect on tubular homeostasis. Among them, the most significant was BARS (BFA-dependent ADP-Ribosylation Substrate), a protein previously implicated in the maintenance of Golgi architecture. BARS has been found to cause the selective breakdown of the tubular part of the Golgi complex promoting fission events which convert the tubular structures into clusters of vesicles. This effect correlated with the enzymatic activity of BARS, which acts as an acyl-CoA dependent lysophosphatidic acid acyl transferase (LPAAT), increasing phosphatidic acid (PA) levels in Golgi membranes. This suggests that local modifications of the composition of the lipid bilayer is a possible mechanism for the fission of membranous tubules.

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