Stacks on tracks

Targeting GFP to the plant Golgi apparatus

Our work on in vivo imaging of the plant endomembrane system started in 1996 with the successful targeting of GFP to the plant Golgi apparatus using a viral expression system
our work started here













In order to further our understanding of the distribution and dynamics of the individual cisternal stacks comprising the Golgi apparatus in plant cells, we spliced the jelly fish green fluorescent protein (GFP) to two proteins that would be predicted to be targeted to Golgi membranes.

These fusions were expressed in leaves of Nicotiana clevlandii using the potato virus X expression system1. Firstly, GFP was fused to the C-terminus of the trans-membrane domain (TMD) of a rat sialyl transferase which would be predicted (in mammalian cells) to target to the trans-region of the Golgi stack. Secondly, GFP was spliced to the C-terminus of the Arabidopsis homologue of the yeast HDEL receptor, AtERD2. This protein acts as a receptor, returning any escaped luminal ER processing proteins from the cis- Golgi back to the ER.

It would therefore be predicted to reside predominantly in the Golgi and cycle to and from the ER. Segments of infected leaves were observed by confocal microscopy and movies made using Zeiss time resolved software. The relationship between the Golgi apparatus, ER and the cytoskeleton was observed in aERD2-GFP expressing leaf cells after paraformaldehyde fixation, permeabilisation and staining with rhodamine conjugated phalloidin.

Results:


Leaf cells expressing the ST-TMD-GFP fusion exhibited a population of highly fluorescent mobile organelles located in the cortical cytoplasm of epidermal cells, the dynamics of which can be viewed in a movie showing targeting of sialyl-transferase signal-anchor sequence to Golgi (8.5MB avi file)

sttmdsmall
Golgi bodies in a tobacco leaf epidermal cell
 

Immunogold electron microscopy with an anti-GFP serum (Molecular Probes) showed the construct to be predominantly located towards the trans-face of the Golgi stacks. This result indicate that the mechanism employed by mammalian cells for locating Golgi transferases also functions in plant cells, suggesting the evolution of a pan kingdom targeting strategy for such processing enzymes. Epidermal leaf cells expressing the aERD2-GFP protein again exhibited a population of highly flourescent mobile organelles, but also a cortical reticular network of ER extending into trans-vacuolar strands.

We have previously described this cortical ER network which can be revealed by GFP-HDEL fusions. Immuno-electron microscopy showed the location of the ERD2-GFP protein was also in the Golgi stacks which as in the TMD-GFP fusion were again highly mobile but now could be observed to be exclusively assocated with the ER and travelled along the cortical ribbons of ER membrane whilst occasionally jumping onto rapidly streaming strands of ER.

golgi on ER

Golgi bodies on the cortical ER network of a tobacco leaf
epidermal cell

This movement of Golgi is both uni- and bi-directional along a strand of ER and often saltatory for several seconds before accelerating down a strand. This novel close association of Golgi and ER can be viewed in the movies- showing tracking of Golgi on cortical ER. aERD2-GFP fusion.


Rhodamine phalloidin staining of the epidermal cells revealed a cortical network of actin filaments which were overlaid by the cortical ER network. By reducing the contrast of the ER in green image of these double labelled preparations it could be seen that the Golgi bodies were also associated with this actin network.

erd3rg2small
Golgi on the actin network of a leaf epidermal cell

 

References:

  • Boevink, P, Oparka, K, Santa Cruz, S, Martin, B, Betteridge, A & Hawes, C R (1998). Stacks on Tracks: the plant Golgi apparatus traffics on an actin/ER network. Plant Journal15, 441-447. Pdficon
  • Boevink, P, Martin, B, Oparka, K, Santa Cruz, S & Hawes, C (1999). Transport of virally expressed green fluorescent protein through the secretory pathway in tobacco leaves is inhibited by cold shock and brefeldin A.Planta208, 392-400.Pdficon