In Vitro Transport on Cis and Trans Sides of the Golgi Involves Two Distinct Types of Coatomer and ADP-ribosylation Factor-independent Transport Intermediates

Abstract

The cisternal maturation model proposes that secretory proteins transit the Golgi in cisternae that mature by the continuous retrograde transport of Golgi enzymes in vesicles. We have tested the hypothesis that de novo generation of transport intermediates containing medial, trans, and trans Golgi network (TGN) enzymes is reconstituted in vitro. Our analysis shows that the majority of transport is mediated by a steady state of transport intermediate production and consumption by Golgi cisternae, with only a minor contribution of pre-existing transport intermediates. Transport in the medial and trans regions of the stack involved intermediates containing Golgi enzymes, apparently moving in a retrograde direction. In contrast, transport between the trans Golgi and TGN was exclusively mediated by intermediates containing secretory protein, as expected for anterograde transport. These intermediates may be physiologically relevant, because only these two specific types of intermediates can be detected in cell homogenates. By analogy to the coatomer (COPI)-independent transport of Golgi enzymes to the endoplasmic reticulum, the steady-state production of intra-Golgi transport intermediates was not impaired by inhibition of COPI vesicle formation. These data suggest a model for COPI-independent intra-Golgi transport by cisternal maturation with a shift in mechanism to anterograde transport at the trans Golgi and TGN boundary.

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

“…This harsh treatment can result in the breakdown of perforated zones enriched in Golgi enzymes into small membrane fragments (Balch et al, 1984). Finally, Pullikuth and Weidman (2002) showed that the release of N-acetylglucosamine transferase and GalT into the supernatant, which apparently contained small membrane fragments, occurred in a COPIindependent manner during an incubation of isolated Golgi membranes with cytosol. Therefore, we propose that these fusion-competent, low-density membrane fractions containing the transferases are formed from perforated areas of cisternae.…”