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

Abstract

In yeast, particular emphasis has been given to endoplasmic reticulum (ER)-derived, cisternal maturation models of Golgi assembly while in mammalian cells more emphasis has been given to golgins as a potentially stable assembly framework. In the case of de novo Golgi formation from the ER after brefeldin A/H89 washout in HeLa cells, we found that scattered, golgin-enriched, structures formed early and contained golgins including giantin, ranging across the entire cis to trans spectrum of the Golgi apparatus. These structures were incompetent in VSV-G cargo transport. Second, we compared Golgi competence in cargo transport to the kinetics of addition of various glycosyltransferases and glycosidases into nascent, golgin-enriched structures after drug washout. Enzyme accumulation was sequential with trans and then medial glycosyltransferases/glycosidases found in the scattered, nascent Golgi. Involvement in cargo transport preceded full accumulation of enzymes or GPP130 into nascent Golgi. Third, during mitosis, we found that the formation of a golgin-positive acceptor compartment in early telophase preceded the accumulation of a Golgi glycosyltransferase in nascent Golgi structures. We conclude that during mammalian Golgi assembly components fit into a dynamic, firstformed, multigolgin-enriched framework that is initially cargo transport incompetent. Resumption of cargo transport precedes full Golgi assembly. INTRODUCTIONDespite its key importance as the central organelle within the secretory pathway, Golgi apparatus assembly remains poorly understood (for a general review, see Shorter and Warren, 2002). The problem is complex and has been studied in several different systems. In yeast, attention has been drawn repeatedly to the relationship between the Golgi apparatus and the endoplasmic reticulum (ER). In particular, in Pichia pastoris, evidence has been presented for the de novo formation of individual Golgi apparatus membrane stacks in association with discrete ER exit sites (Bevis et al., 2002). In Saccharomyces cerevisiae, where ER exit is not organized into discrete export zones, evidence has been presented for the ER derivation of Golgi apparatus membranes in the newly formed bud (Reinke et al., 2004). The appearance of a late Golgi marker in the bud was dependent on the appearance of an early Golgi marker, consistent with the expectations of a cisternal progression/maturation model. In mammalian cells, in contrast, more emphasis has been given to the potential role of Golgi membrane clusters and Golgi matrix proteins in organizing Golgi assembly. Golgi matrix proteins belong to the golgin family of coiled-coiledrich proteins and are often, but not always, e.g., giantin, peripherally associated with Golgi apparatus membranes (for review, see Short et al., 2005). These proteins may serve a structural role in organizing Golgi apparatus membranes and subcompartments and as such may have a membrane organizing role in both mitotic and de novo mammalian Golgi assembly.In mammalian cells, the long recognized exam...

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

“…Next, we compared the distribution of GM130 with that of Bap31 during recovery from BFA treatment. 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).…”

“…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). This is perhaps due to a relatively weak action of H89 on COS-7 cells, as described above.…”

“…We could not test H89 at concentrations higher than 100 M because COS-7 cells were detached from coverslips, even those coated with poly-l-lysine. It seemed that the effect of H89 on COPII localization is weaker in COS-7 cells than HeLa cells (Lee and Linstedt, 2000;Puri and Linstedt, 2003;Jiang et al, 2006). Next, we examined whether Bap31, upon H89 treatment, moves to the juxtanuclear region through ER exit sites, as in the case of secretory cargos.…”

Microtubule-independent secretion requires functional maturation of Golgi elements

“…In addition, removal of microtubules does not prevent retrograde transport of Golgi proteins to the ER. This was well demonstrated by preventing ER export (using BFA or H89, or dominant-negative Sar1) at the same time as microtubule disruption (Jiang et al, 2006;Puri and Linstedt, 2003;Storrie et al, 1998). In our study, inhibiting new synthesis of proteins did not prevent functional maturation of Golgi elements.…”

Rab33b and Rab6 are Functionally Overlapping Regulators of Golgi Homeostasis and Trafficking

“…• C in the presence of cycloheximide to chase newly synthesized tsO45G into the Golgi apparatus and plasma membrane (23,49). Following deconvolution with HUYGENS PROFESSIONAL software (Scientific Volume Imaging), tsO45G-GFP distribution patterns were scored visually as previously described (31).…”