Submitted on June 13, 2007
Accepted on September 28, 2007
Activation of ADP-ribosylation factor (Arf) regulates biogenesis of the
ATP7A containing trans-Golgi network compartment and its Cu-induced
trafficking
Zoe Gail Holloway1, Robert Grabski2, Tomasz Szul2, Melanie Styers2, Julie Coventry1, Anthony P Monaco1, and ELIZABETH SZTUL2*
1 WTCHG, University of Oxford, Oxford, United Kingdom
2 CELL BIOLOGY, UNIVERSITY OF ALABAMA AT BIRMINGHAM, BIRMINGHAM, Alabama, United States
* To whom correspondence should be addressed. E-mail: esztul{at}uab.edu.
ATP7A (MNK) regulates copper homeostasis by translocating from a compartment localized within the trans-Golgi network (TGN) to the plasma membrane (PM) in response to increased copper load. The mechanisms that regulate the biogenesis of the MNK compartment and the trafficking of MNK are unclear. Here, we show the architecture of the MNK compartment is linked to the structure of the Golgi ribbon. Depletion of p115 tethering factor, which causes fragmentation of the Golgi ribbon, also disrupts the MNK compartment. In p115-depleted cells, MNK localizes to punctate structures that pattern on Golgi mini-stacks dispersed throughout the cell. Despite altered localization, MNK trafficking still occurs, and MNK relocates from and returns to the fragmented compartment in response to copper. We further show the biogenesis of the MNK compartment requires activation of Arf1 GTPase, shown previously to facilitate the biogenesis of the Golgi ribbon. Preventing activation of cellular Arf1 by (1) expressing an inactive "empty" form of Arf (Arf1/N126I), (2) expressing an inactive form of GBF1 (GBF1/E794K), guanine nucleotide exchange factor for Arf1, or (3) treating cells with Brefeldin A (BFA), an inhibitor of GBF1, disrupts MNK into a diffuse pattern. Importantly, preventing Arf activation inhibits copper-responsive trafficking of MNK to the PM. Our findings support a model in which active Arf is essential for the generation of the MNK compartment and for copper-responsive trafficking of MNK from here to the PM. Our findings provide an exciting foundation for identifying Arf1 effectors that facilitate the biogenesis of the MNK compartment and MNK traffic.
