p53 acts as a tumor suppressor by inducing both growth arrest and apoptosis. p53-induced apoptosis can occur without new RNA synthesis through an unknown mechanism. In human vascular smooth muscle cells, p53 activation transiently increased surface Fas (CD95) expression by transport from the Golgi complex. Golgi disruption blocked both p53-induced surface Fas expression and apoptosis. p53 also induced Fas-FADD binding and transiently sensitized cells to Fas-induced apoptosis. In contrast, lpr and gld fibroblasts were resistant to p53-induced apoptosis. Thus, p53 can mediate apoptosis through Fas transport from cytoplasmic stores.

Myosin VI plays a role in the maintenance of Golgi morphology and in exocytosis. In a yeast 2-hybrid screen we identified optineurin as a binding partner for myosin VI at the Golgi complex and confirmed this interaction in a range of protein interaction studies. Both proteins colocalize at the Golgi complex and in vesicles at the plasma membrane. When optineurin is depleted from cells using RNA interference, myosin VI is lost from the Golgi complex, the Golgi is fragmented and exocytosis of vesicular stomatitis virus G-protein to the plasma membrane is dramatically reduced. Two further binding partners for optineurin have been identified: huntingtin and Rab8. We show that myosin VI and Rab8 colocalize around the Golgi complex and in vesicles at the plasma membrane and overexpression of constitutively active Rab8-Q67L recruits myosin VI onto Rab8-positive structures. These results show that optineurin links myosin VI to the Golgi complex and plays a central role in Golgi ribbon formation and exocytosis.

We have investigated the requirement for Ca2+ in the fusion and content mixing of rat hepatocyte late endosomes and lysosomes in a cell-free system. Fusion to form hybrid organelles was inhibited by 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA), but not by EGTA, and this inhibition was reversed by adding additional Ca2+. Fusion was also inhibited by methyl ester of EGTA (EGTA-AM), a membrane permeable, hydrolyzable ester of EGTA, and pretreatment of organelles with EGTA-AM showed that the chelation of lumenal Ca2+ reduced the amount of fusion. The requirement for Ca2+ for fusion was a later event than the requirement for a rab protein since the system became resistant to inhibition by GDP dissociation inhibitor at earlier times than it became resistant to BAPTA. We have developed a cell-free assay to study the reformation of lysosomes from late endosome–lysosome hybrid organelles that were isolated from the rat liver. The recovery of electron dense lysosomes was shown to require ATP and was inhibited by bafilomycin and EGTA-AM. The data support a model in which endocytosed Ca2+ plays a role in the fusion of late endosomes and lysosomes, the reformation of lysosomes, and the dynamic equilibrium of organelles in the late endocytic pathway.