Abstract
The plant Golgi apparatus modifies and sorts incoming proteins from the endoplasmic reticulum (ER) and synthesizes cell wall matrix material. Plant cells possess numerous motile Golgi bodies, which are connected to the ER by yet to be identified tethering factors. Previous studies indicated a role for cis-Golgi plant golgins, which are long coiled-coil domain proteins anchored to Golgi membranes, in Golgi biogenesis. Here we show a tethering role for the golgin AtCASP at the ER-Golgi interface. Using live-cell imaging, Golgi body dynamics were compared in Arabidopsis thaliana leaf epidermal cells expressing fluorescently tagged AtCASP, a truncated AtCASP-ΔCC lacking the coiled-coil domains, and the Golgi marker STtmd. Golgi body speed and displacement were significantly reduced in AtCASP-ΔCC lines. Using a dual-colour optical trapping system and a TIRF-tweezer system, individual Golgi bodies were captured in planta. Golgi bodies in AtCASP-ΔCC lines were easier to trap and the ER-Golgi connection was more easily disrupted. Occasionally, the ER tubule followed a trapped Golgi body with a gap, indicating the presence of other tethering factors. Our work confirms that the intimate ER-Golgi association can be disrupted or weakened by expression of truncated AtCASP-ΔCC and suggests that this connection is most likely maintained by a golgin-mediated tethering complex.
Highlights
The architecture of the Golgi apparatus is distinct and seemingly simple
COPII-coated membrane carriers function in anterograde endoplasmic reticulum (ER)-to-Golgi transport, whereas COPI-coated vesicles transport proteins backwards within the stack and from the cis-Golgi stack back to the ER for recycling (Robinson et al, 2015)
If AtCASP played a role in tethering events between the ER and Golgi bodies, deleting its coiled-coil domain would change its mechanical properties and as such could be predicted to affect Golgi morphology, function or dynamics, possibly resulting in changes in: (i) the subcellular location of the fluorescent mutant compared to the full-length protein, (ii) the subcellular location of Golgi bodies in relation to the ER, (iii) Golgi body dynamics, such as speed or displacement, or (iv) the physical interaction between Golgi bodies and the ER tested by optical tweezer based displacement of Golgi bodies
Summary
The architecture of the Golgi apparatus is distinct and seemingly simple. It is an organelle composed of lipids and proteins, arranged as a polarized stack of flattened cisternae, capable of processing and distributing secretory cargo around and out of the cell (Staehelin and Moore, 1995; Polishchuk and Mironov, 2004; Klumperman, 2011). The exact mechanisms of Golgi stack assembly and maintenance are still not fully understood (Wang and Seemann, 2011). It is Abbreviations: ER, endoplasmic reticulum; ERES, ER exit sites; mRFP, monomeric red fluorescent protein. COPII-coated membrane carriers function in anterograde ER-to-Golgi transport, whereas COPI-coated vesicles transport proteins backwards within the stack and from the cis-Golgi stack back to the ER for recycling (Robinson et al, 2015)
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