Maintenance of Golgi structure and function depends on the integrity of ER export

Abstract

The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells. Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle. We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model. Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm. When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1[T31N]), the Golgi structure disassembles, leaving no residual Golgi membranes. Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos. A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region. In cells expressing Sar1[T39N], a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER. These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis. Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1–COPII and Arf1–coatomer systems.

Bap31 Is an Itinerant Protein That Moves between the Peripheral Endoplasmic Reticulum (ER) and a Juxtanuclear Compartment Related to ER-associated Degradation

“…In HeLa cells, Bap31 cycled between the juxtanuclear and peripheral regions more slowly compared with COS-7 cells; the ratio of HeLa cells with juxtanuclear Bap31 accumulation to total cells was maximum at 120 min after BFA washout (Supplemental Figure S5). As shown previously (Nakamura et al, 1995;Ward et al, 2001) and in this study ( Figure 1E), BFA treatment caused some dispersion of ERGIC-53-positive clusters at the juxtanuclear region without changing punctate ERGIC-53 staining at the peripheral region. When BFA was washed out, ERGIC-53 seemed to become concentrated at the juxtanuclear region (Supplemental Figure S6).…”

“…GM130 is one of the fast-moving proteins during BFA recovery (Jiang et al, 2006). As reported previously (Nakamura et al, 1995;Seemann et al, 2000;Ward et al, 2001), GM130 was distributed in punctate structures in BFA-treated cells (Supplemental Figure S3, second row). Although previous studies showed that combined treatment of normal rat kidney or HeLa cells with BFA followed by BFA plus H89 causes ER localization of GM130 (Puri and Linstedt, 2003;Jiang et al, 2006), such redistribution was not observed in COS-7 cells when 50 M H89 in addition to 10 M BFA was used (data not shown).…”

“…Expression of these mutants in cells block ER-Golgi transport at specific stages in conventional transport processes (Ward et al, 2001;Altan-Bonnet et al, 2003). When inactive Sar1p (Sar1p[T39N]) or the active form (Sar1p[H79G]) was expressed in COS-7 cells, H89-induced juxtanuclear accumulation of Bap31 was blocked ( Figure 4A).…”

Poliovirus Protein 3A Binds and Deregulates LIS1, Causing Block of Membrane Protein Trafficking and Deregulation of Cell Division

“…LIS1 was previously shown to be involved in the dynein motor complex that determines microtubule-associated movement of organelles and membrane vesicles [18][19][20][21][22][23][24][25][26] and maintenance of the integrity of the Golgi apparatus and juxta-centrosomal distribution of its membranes (reviewed in refs. [27][28][29][30][31][32][33][34][35]. The comparison of the effects of 3A and dominant negative mutants of LIS1, which inhibit surface receptor recovery, suggests that 3A acts by trapping LIS1-containing complexes on the cytoplasmic surface of the ER, thereby preventing it from transporting proteins between the ER and the Golgi.…”

Rapid, Endoplasmic Reticulum-independent Diffusion of the Mitotic Golgi Haze

“…This might help explain the variability in the amount of Golgi haze that is seen in different cell types. The status of the matrix is, however, still unclear, and its existence has been disputed (Zaal et al, 1999;Miles et al, 2001;Ward et al, 2001;Stroud et al, 2003). In the merged ER-Golgi model of partitioning, the MGCs are viewed as intermediates on the pathway leading to merger of the Golgi and the ER.…”