Abstract
Herein, we report the stepwise transport of multiple plant Golgi membrane markers during disassembly of the Golgi apparatus in tobacco leaf epidermal cells in response to the induced expression of the GTP-locked Sar1p or Brefeldin A (BFA), and reassembly on BFA washout. The distribution of fluorescent Golgi-resident N-glycan processing enzymes and matrix proteins (golgins) with specific cis–trans-Golgi sub-locations was followed by confocal microscopy during disassembly and reassembly. The first event during Golgi disassembly was the loss of trans-Golgi enzymes and golgins from Golgi membranes, followed by a sequential redistribution of medial and cis-Golgi enzymes into the endoplasmic reticulum (ER), whilst golgins were relocated to the ER or cytoplasm. This event was confirmed by fractionation and immuno-blotting. The sequential redistribution of Golgi components in a trans–cis sequence may highlight a novel retrograde trafficking pathway between the trans-Golgi and the ER in plants. Release of Golgi markers from the ER upon BFA washout occurred in the opposite sequence, with cis-matrix proteins labelling Golgi-like structures before cis/medial enzymes. Trans-enzyme location was preceded by trans-matrix proteins being recruited back to Golgi membranes. Our results show that Golgi disassembly and reassembly occur in a highly ordered fashion in plants.
Highlights
The plant Golgi apparatus has a unique architecture and is organized as polarized stacks of flattened cisternae that exhibit a structural and functional cis-to-trans polarity [1,2]
In a recent study of Golgi membrane dynamics in tobacco leaf epidermal cells we showed that the redistribution of the two cis/medial-Golgi matrix proteins AtCASP and GC1 (Golgin Candidate 1, an Arabidopsis golgin-84 isoform) to the endoplasmic reticulum (ER) and cytoplasm, respectively, was preceded by the relocation of the trans-Golgi membrane marker ST after Brefeldin A (BFA) treatment and after induction of Sar1-GTP expression [30]
N-glycan processing enzymes define distinct regions of the Golgi apparatus in plants To study the fate of individual cisternae during Golgi disassembly and reassembly we used fluorescent protein-tagged integral Golgi-resident N-glycan processing enzymes, which are differentially localized in sequential Golgi cisternae according to their position in the biosynthetic pathway
Summary
The plant Golgi apparatus has a unique architecture and is organized as polarized stacks of flattened cisternae that exhibit a structural and functional cis-to-trans polarity [1,2]. More evidence in favour of recycling comes from the over-expression of a GTPlocked Sar1p, the small GTPase which initiates COPII coat formation at ERES, leading to a block in ER exit, as well as the addition of Brefeldin A (BFA), which blocks assembly of COPI vesicles [24] Both perturbations induce disassembly of Golgi cisternae and redistribution of Golgi membrane markers into the nearby ER network [4,23,25,26,27,28,29,30], an event similar to that reported for mammalian cells [21,31,32,33,34,35].
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