Dynamic Nucleation of Golgi Apparatus Assembly from the Endoplasmic Reticulum in Interphase HeLa Cells

Abstract

Models of Golgi apparatus biogenesis and maintenance are focused on two possibilities: one is self‐assembly from the endoplasmic reticulum, and the other is nucleation by a stable template. Here, we asked in three different experimental situations whether assembly of the Golgi apparatus might be dynamically nucleated. During microtubule depolymerization, the integral membrane protein p27 and the peripheral Golgi protein GM130, appeared in newly formed, scattered Golgi elements before three different Golgi apparatus cisternal enzymes, whereas GRASP55, a medial peripheral Golgi protein, showed, if anything, a tendency to accumulate in scattered Golgi elements later than a cisternal enzyme. During Golgi formation after brefeldin A washout, endoplasmic reticulum exit of Golgi resident enzymes could be completely separated from that of p27 and GM130. p27 and GM130 accumulation was onto newly organized perinuclear structures, not brefeldin A remnants, and preceded that of a cisternal enzyme. Reassembly was completely sensitive to guanosine 5′‐diphosphate‐restricted Sar1p. When cells were microinjected with Sar1pWT DNA to reverse a guanosine 5′‐diphosphate‐restricted Sar1p endoplasmic reticulum‐exit block phenotype, GM130 and p27 collected perinuclearly with little to no exit of a cisternal enzyme from the endoplasmic reticulum. The overall data strongly indicate that the assembly of the Golgi apparatus can be nucleated dynamically by GM130/p27 associated structures. We define dynamic nucleation as the first step in a staged organelle assembly process in which new component association forms a microscopically visible structure onto which other components add later, e.g. Golgi cisternae.

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

“…In sum, Golgi assembly is slow at 32°C with both cis-and trans-golgins accumulating more rapidly into post-ER punctate structures than a Golgi glycosyltransferase. The slow accumulation of GalNAcT2 into post-ER structures is consistent with data trends reported earlier at higher temperatures (Puri and Linstedt, 2003;Kasap et al, 2004). Note: the 2-h accumulation of GalNAcT2 into perinuclear patches was consistent with that observed for tsO45G, albeit GPP130 was slow.…”

“…Similarly, Puri and Linstedt (2003) find that GM130 accumulates early in post-ER structures. In previous work, we have shown that a member of the p24 family of transmembrane proteins is an early component of such structures (Kasap et al, 2004). We found in our experiments no evidence for a bolus of Golgi proteins and lipids emerging as a group from the ER.…”

“…By extrapolation, de novo Golgi formation from the ER is predicted. Experimental evidence for de novo Golgi formation from the ER comes both from work in the yeast P. pastoris (Bevis et al, 2002) and S. cerevisiae (Reinke et al, 2004) and with mammalian cells (Puri and Linstedt, 2003;Kasap et al, 2004). Much of the mammalian cell work has examined Golgi formation either at scattered sites during microtubule depolymerization or after washout of the drugs BFA or BFAϩH89, treatments that disperse Golgi proteins to the ER (Lippincott-Schwartz et al, 1989;Puri and Linstedt, 2003).…”

“…The combined effect of BFA and H89 treatment is to redistribute both Golgi transmembrane proteins and golgins to the ER from which de novo assembly occurs (Puri and Linstedt, 2003). The first detectable step in de novo Golgi assembly is the formation of a post-ER structure positive for the golgin GM130 and cargo receptor gp27 (Puri and Linstedt, 2003;Kasap et al, 2004). This golgin-positive nascent Golgi structure appears to have an acceptor role in subsequent complete Golgi apparatus assembly.…”

Ordered assembly of the duplicating Golgi in Trypanosoma brucei

“…Studies have, therefore, been restricted to analysis of Golgi reassembly after treatment with drugs such as Brefeldin A (11)(12)(13)(14), although it is not clear to what extent this is a suitable model for biogenesis.…”