The Role of ARF1 and Rab GTPases in Polarization of the Golgi Stack

Abstract

The organization and sorting of proteins within the Golgi stack to establish and maintain its cis to trans polarization remains an enigma. The function of Golgi compartments involves coat assemblages that facilitate vesicle traffic, Rab-tether-SNAP receptor (SNARE) machineries that dictate membrane identity, as well as matrix components that maintain structure. We have investigated how the Golgi complex achieves compartmentalization in response to a key component of the coat complex I (COPI) coat assembly pathway, the ARF1 GTPase, in relationship to GTPases-regulating endoplasmic reticulum (ER) exit (Sar1) and targeting fusion (Rab1). Following collapse of the Golgi into the ER in response to inhibition of activation of ARF1 by Brefeldin A, we found that Sar1-and Rab1-dependent Golgi reformation took place at multiple peripheral and perinuclear ER exit sites. These rapidly converged into immature Golgi that appeared as onion-like structures composed of multiple concentrically arrayed cisternae of mixed enzyme composition. During clustering to the perinuclear region, Golgi enzymes were sorted to achieve the degree of polarization within the stack found in mature Golgi. Surprisingly, we found that sorting of Golgi enzymes into their subcompartments was insensitive to the dominant negative GTP-restricted ARF1 mutant, a potent inhibitor of COPI coat disassembly and vesicular traffic. We suggest that a COPI-independent, Rab-dependent mechanism is involved in the rapid reorganization of resident enzymes within the Golgi stack following synchronized release from the ER, suggesting an important role for Rab hubs in directing Golgi polarization.

Golgi structural stability and biogenesis depend on associated PKA activity

“…The time of association, however, was clearly delayed in comparison to the time of re-emergence from the ER. In agreement with previous studies (Bannykh et al, 2005;Puri and Linstedt, 2003), Golgi membranes exited the ER at multiple peripheral sites that with time converged to the juxtanuclear region where they fused and reconstructed the ribbon organization. PKA association was coincident with ribbon formation.…”

“…These elements corresponded to globular Golgi stacks composed of multiple concentrically arranged cisternae. Interestingly, similar structures have been recently reported to occur in cells recovering from BFA treatment in the absence of any other additional agent (Bannykh et al, 2005). This suggests that they might represent transient intermediates in the normal process of Golgi reassembly from the ER.…”

“…Bars, 10 m. (B) Non-transfected cells were incubated with 2 g/ml BFA for 1 hour at 37°C before fixation and processing for electron microscopy. A vesicular-tubular cluster believed to represent the starting structure for Golgi reassembly (Bannykh et al, 2005) is shown. (C) Cells were allowed to recover from BFA treatment for 3 hours at 37°C in the continuous presence of 20 M H89 before fixation and processing.…”

Capacity of the Golgi Apparatus for Cargo Transport Prior to Complete Assembly

“…A similar order was observed at 37°C. Recent results suggest that COPI coat protein-dependent retrieval mechanisms are unlikely to play an important role in achieving polarity within the Golgi cisternal stack (Bannykh et al, 2005). Our results indicate that a polarized framework is present early during Golgi apparatus assembly.…”

“…We treated briefly with BFA or BFAϩH89 in order to disperse Golgi proteins and lipids into the ER. Recent work indicates that with longer BFA treatment there is a sorting of Golgi proteins and lipids into an ER exit site proximal structure referred to as vesicular tubular cluster or VTC by some (Bannykh et al, 2005;Marondes et al, 2006). The identification of the VTC is supported by electron microscopy.…”