Golgi Enzymes Are Enriched in Perforated Zones of Golgi Cisternae but Are Depleted in COPI Vesicles

Abstract

In the most widely accepted version of the cisternal maturation/progression model of intra-Golgi transport, the polarity of the Golgi complex is maintained by retrograde transport of Golgi enzymes in COPI-coated vesicles. By analyzing enzyme localization in relation to the three-dimensional ultrastructure of the Golgi complex, we now observe that Golgi enzymes are depleted in COPI-coated buds and 50-to 60-nm COPI-dependent vesicles in a variety of different cell types. Instead, we find that Golgi enzymes are concentrated in the perforated zones of cisternal rims both in vivo and in a cell-free system. This lateral segregation of Golgi enzymes is detectable in some stacks during steady-state transport, but it was significantly prominent after blocking endoplasmic reticulum-to-Golgi transport. Delivery of transport carriers to the Golgi after the release of a transport block leads to a diminution in Golgi enzyme concentrations in perforated zones of cisternae. The exclusion of Golgi enzymes from COPI vesicles and their transport-dependent accumulation in perforated zones argues against the current vesicle-mediated version of the cisternal maturation/progression model.

Analogs of the Golgi complex in microsporidia: structure and avesicular mechanisms of function

“…If microsporidia can transport cargo without the use of COP-I and COP-II vesicles, this means that this possibility should be tested for in mammalian cells. Our analysis shows that there are no strong results that consistently demonstrate that COP-I and COP-II vesicles are transport carriers (see Mironov et al, 2003;Mironov et al, 2005;Kweon et al, 2004). At this stage, it is preliminary to state that COP-I and COP-II vesicles are not important for intracellular transport.…”

“…However, despite this, we reasoned that if coatomer-dependent vesicles normally form but fuse immediately with the acceptor compartment, they should accumulate, and hence become detectable, if fusion is inhibited. The protein NSF is essential in the known pathways of Golgi membrane fusion (including fusion of COP-I vesicles to Golgi membranes) and it is present in the microsporidia genome (Katinka et al, 2001); the highly membranepermeable reagent N-ethylmaleimide (NEM) under controlled conditions Kweon et al, 2004) can relatively selectively inactivate NSF. Fresh homogenates of fat bodies of infected Gryllus bimaculatus crickets were thus treated with NEM for 15 minutes on ice, and they were fixed 15 minutes after wash-out of the NEM, with dithiothreitol (DTT).…”

“…This model would require the Golgi enzymes to be enriched in these vesicles. However, our own recent study (Kweon et al, 2004) based on several complementary approaches showed that Golgi enzymes are depleted in actual COP-I-dependent vesicles. Furthermore, there are no COP-I-coated buds detectable on the last transcisternae (Ladinsky et al, 1999;Ladinsky et al, 2002) although some Golgi enzymes, such as sialyltransferase and fucosyltransferase, are present therein (Rabouille et al, 1995;Opat et al, 2001).…”

A role for the host coatomer and KDEL receptor in early vaccinia biogenesis

“…ImmunoGold EM examination of cryosections was performed essentially as described (29). Specificity of the antibodies used for ImmunoGold labeling has been established for ␤-COP (29) and KDELR1-myc (24).…”

Mutations in a Highly Conserved Region of the Arf1p ActivatorGEA2Block Anterograde Golgi Transport but Not COPI Recruitment to Membranes

“…There is evidence in both yeast and mammalian cells that COPI mediates the retrograde trafficking of glycosylation enzymes, cargo receptors, and fusion machinery (Nakano, 2004). The precise mechanism of this retrograde trafficking is a subject of ongoing research, and there is debate as to whether retrograde-directed glycosylation enzymes in mammalian cells are packaged into COPI vesicles or are sorted into tubules that connect Golgi cisternae (Kweon et al, 2004;Trucco et al, 2004). Trucco et al (2004) have proposed that COPI vesicles contain not Golgi enzymes but primarily the fusion machinery, such as the SNARE protein membrin.…”