Sequential depletion and acquisition of proteins during Golgi stack disassembly and reformation

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.

Figure 1

GnTI-mRFP, GMII-CFP and GALT1-GFP show distinct intra-Golgi distributions. Confocal images showing GnTI-mRFP, GMII-CFP and GALT1-GFP transiently expressed alone (A–C) or in pairs (D–F) in wild-type tobacco leaf epidermal cells. Double-colour images (D–F) display highly magnified Golgi stacks double-labelled by (D) GnTI-mRFP (magenta) and GMII-CFP (green), (E) GnTI-mRFP (magenta) and GALT1-GFP (green) or (F) GMII-CFP (magenta) and GALT1-GFP (green). Note the shift of the overlapping signals with the central region being white and the non-overlapping regions being green and magenta. Scale bars = 20 µm in (A–C) and 2 µm in (D–F).

Figure 2

GALT1-GFP is redistributed into the ER before GnTI-mRFP during Golgi disassembly. Time–course of the effects of BFA and Sar1-GTP expression, respectively, on the Golgi markers GnTI-mRFP (magenta) and GALT1-GFP (green). The expression of markers was either checked before and after treatment of wild-type tobacco leaves with 100 µg/mL BFA over a time-period of 2–3 h (A–C) or before and after induction of stable inducible tobacco plants with a 20 µg/mL dexamethasone solution over a time-period of 18–25 h (D–F). Double-colour images (a merge of green and magenta channels) were obtained by confocal microscopy at different time-points of each experiment. Scale bars = 10 µm.

Figure 3

Golgi disassembly occurs in a trans-to-cis direction. Confocal images showing GnTI-mRFP (magenta), GMII-CFP (cyan) and GALT1-GFP (green) transiently expressed together in leaves of stable Sar1-GTP-inducible tobacco plants before (A) and 23.5 h (B) after treatment with dexamethasone (20 µg/mL). The inset (A) shows a magnification of the shift of overlapping signals within triple-labelled Golgi stacks. The arrow (B) indicates a Golgi stack solely labelled by GMII-CFP, whereas arrowheads indicate stacks labelled only by GnTI-mRFP. Scale bars = 5 µm.

Figure 4

Subcellular fractionation analysis of the effect of BFA on GnTI-mRFP and GALT1-GFP expressing cells.N. benthamiana leaf epidermal cells transiently coexpressing GnTI-mRFP and GALT1-GFP were treated with BFA (75 µg/mL) for 45 min. Microsomes were obtained from mock-treated (control, A) and BFA-treated (BFA, B) leaf tissue and separated on a discontinuous sucrose density gradient. Each of the 21 fractions was subjected to immunoblot analysis with antibodies against BiP (ER marker), GFP (GALT1-GFP) and mRFP (GnTI-mRFP). Fractions were numbered from top (fraction 1 in the first lane on the left) to bottom (fraction 21 in the last lane on the right) of the gradient. In order to compare the migration behaviour of each protein after control and BFA treatments, respectively, the intensity of bands on each immunoblot was quantified and expressed as relative intensities (in %, C).

Figure 5

GnTI-mRFP is found at reforming Golgi bodies before GALT1-GFP after BFA washout. Confocal images showing wild-type tobacco leaf epidermal cells coexpressing GnTI-mRFP (magenta) and GALT1-GFP (green) 1.5 h (A) and 3 h (B) after BFA washout (w/o). Before removal of BFA, Golgi proteins were forced back into the ER by a 2-h BFA treatment (100 µg/mL). Scale bars = 10 µm.

Figure 6

GnTI-GFPglyc and GALT1-GFP have similar dynamic behaviours. A and B) Confocal images showing a time series of the fluorescence recovery in photobleached Golgi stacks (circles) in tobacco leaf epidermal cells transiently expressing either GnTI-GFPglyc (A) or GALT1-GFP (B). Before bleaching, cells were treated for 1 h with latrunculin B (25 µm). Time is expressed in seconds (s) at the bottom left of frames. Scale bars = 5 µm. (C) Fluorescence recovery graph: measured, normalized and fitted FRAP plotted against time. Values for maximum fluorescence recovery (MFR), t_1/2 (fluorescence half-time of recovery) and curve fit r² were derived from these data and are summarized in (D).

Figure 7

The effects of BFA addition and washout on the cis/medial-Golgi matrix protein mRFP-AtCASP and the trans-Golgi marker GALT1-GFP. Confocal images showing BFA treatment and washout (w/o) in wild-type tobacco leaf epidermal cells coexpressing GALT1-GFP (green) and mRFP-AtCASP (magenta). Scale bars = 10 µm. (A) Before BFA addition; (B) 1 h after BFA addition; (C) 1.5 h after BFA washout; (D) 3 h after BFA washout.

Figure 8

During Golgi reconstruction the cis/medial-Golgi marker GnTI-mRFP recovers before the trans-Golgi matrix protein GFP-GC5. Confocal images showing the BFA treatment and washout (w/o) in wild-type tobacco leaf epidermal cells coexpressing GnTI-mRFP (magenta) and GFP-GC5 (green). Scale bars = 5 µm. (A) Before BFA addition; (B) 1 h after BFA addition; (C) 30 min after BFA washout; (D) 4.5 h after BFA washout.

Figure 9

The effects of BFA addition and washout on the trans-Golgi matrix protein GFP-GC5 and the trans-Golgi marker ST-mRFP. Confocal images showing the BFA treatment and washout (w/o) in wild-type tobacco leaf epidermal cells coexpressing GFP-GC5 (green) and ST-mRFP (magenta). Scale bars = 10 µm. (A) Before BFA treatment; (B) 30 min after BFA addition; (C) 15 min after BFA washout; (D) 4.5 h after BFA washout. (E) Diagram showing a quantification of Golgi bodies labelled either by GFP-GC5 and ST-mRFP or solely GFP-GC5 during BFA treatment and washout.

Figure 10

De novo assembly of cis-Golgi cisternae is preceded by structures positive for the cis/medial-Golgi matrix protein GFP-GC1. Confocal images showing the BFA treatment and washout (w/o) in wild-type tobacco leaf epidermal cells coexpressing GFP-GC1 (green) and GnTI-mRFP (magenta). Scale bars = 10 µm. (A) Before BFA addition; (B) 1.5 h after BFA addition; (C) 1.5 h after BFA washout; (D) 3.5 h after BFA washout.