Abstract
Multisubunit members of the CATCHR family: COG and NRZ complexes, mediate intra-Golgi and Golgi to ER vesicle tethering, respectively. We systematically addressed the genetic and functional interrelationships between Rabs, Kifs, and the retrograde CATCHR family proteins: COG3 and ZW10, which are necessary to maintain the organization of the Golgi complex. We scored the ability of siRNAs targeting 19 Golgi-associated Rab proteins and all 44 human Kifs, microtubule-dependent motor proteins, to suppress CATCHR-dependent Golgi fragmentation in an epistatic fluorescent microscopy-based assay. We found that co-depletion of Rab6A, Rab6A’, Rab27A, Rab39A and two minus-end Kifs, namely KIFC3 and KIF25, suppressed both COG3- and ZW10-depletion-induced Golgi fragmentation. ZW10-dependent Golgi fragmentation was suppressed selectively by a separate set of Rabs: Rab11A, Rab33B and the little characterized Rab29. 10 Kifs were identified as hits in ZW10-depletion-induced Golgi fragmentation, and, in contrast to the double suppressive Kifs, these were predominantly plus-end motors. No Rabs or Kifs selectively suppressed COG3-depletion-induced Golgi fragmentation. Protein-protein interaction network analysis indicated putative direct and indirect links between suppressive Rabs and tether function. Validation of the suppressive hits by EM confirmed a restored organization of the Golgi cisternal stack. Based on these outcomes, we propose a three-way competitive model of Golgi organization in which Rabs, Kifs and tethers modulate sequentially the balance between Golgi-derived vesicle formation, consumption, and off-Golgi transport.
Highlights
In most mammalian cells, the Golgi apparatus is organized into a dynamic, ribbon-like structure, which is generated by laterally linked Golgi stacks consisting of several cisternae aligned in parallel
We demonstrated that 4 Rabs and 2 Kifs suppressed both ZW10- and COG3-depletion-induced Golgi fragmentation (ZDI- and CDI-Golgi fragmentation, respectively) strongly indicating that these two pathways share common initial steps
We initially tested strategies for robust quantitative analysis of the Golgi complex when imaged by fluorescence and electron microscopy in three states: native compact, fragmented and restored after the double knockdown of Rab6 and retrograde Golgi tethers
Summary
The Golgi apparatus is organized into a dynamic, ribbon-like structure, which is generated by laterally linked Golgi stacks consisting of several cisternae aligned in parallel (for review, see Wei and Seemann, 2010). Inhibition/redirection of Golgi membrane trafficking can lead to disruption of the interphase Golgi apparatus with a classic example being the disruption of the Golgi ribbon into scattered mini-stacks due to druginduced microtubule depolymerization (Rogalski et al, 1984; Thyberg and Moskalewski, 1985; Cole et al, 1996; Yang and Storrie, 1998) Another case is retrograde Golgi tether-dependent membrane trafficking in which tether depletion results in organelle scattering. The RNAi depletion of the COG3 protein causes Golgi fragmentation accompanied by accumulation of scattered glycosyltransferase-positive vesicles (Zolov and Lupashin, 2005; Shestakova et al, 2007) Knockdown of yet another CATCHR family tether, NRZ complex, through depletion of ZW10 (centromere/kinetochore protein ZW10 homolog) or RINT-1 (Rad50-interacting protein 1) leads to fragmentation of Golgi ribbon into a cluster of punctate Golgi elements (Hirose et al, 2004; Sun et al, 2007). These outcomes and approach should be generalizable to large scale studies of protein interactions that define spatiotemporal Golgi organization
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