The role of Atgolgin-84A at the plant ER-Golgi interface

The eukaryotic cell is compartmentalised into an endomembrane system organised into several organelles. Some of these organelles, namely endoplasmic reticulum (ER) and Golgi bodies, are part of a so-called secretory pathway where vital cell components travel to reach the correct final destination or are recycled for further processing. In the plant cell cytoplasm there are numerous stacks of membrane bounded cisternae, each of which constitutes a discrete Golgi body. Golgi bodies are responsible in part for the processing of proteins received from the ER and their distribution to the plasma membrane and other compartments. Exactly how this motile structure is maintained while providing vital functions for the cell is still poorly understood. The ER is physically connected to Golgi bodies, and Golgi matrix components, such as golgins, have been identified and suggested to function as putative tethering factors. Golgins are proteins anchored to the Golgi membrane by the C-terminus either through transmembrane domains (TMDs) or interaction with small regulatory GTPases. The golgin N-terminus contains long coiled-coil domains which consist of a number of α-helices wrapped around each other to form a structure similar to a rope being made from several strands, reaching into the cytoplasm. Atgolgin84A may act as tethering factor at the ER-Golgi interface and within the Golgi stack. In animal cells golgins are also implicated in specific recognition of cargo at the Golgi. In plants, there is no clear evidence for the localisation of Atgolgin-84A at the Golgi. To investigate Atgolgin-84A subcellular localisation and putative function as a tether, fluorescent fusions to Atgolgin-84A and Atgolgin-84A truncation lacking the coiled-coil domains (Atgolgin-84A1-557) were transiently expressed in Nicotiana tabacum and imaged by confocal microscopy with Airyscan detector. High-resolution confocal imaging was used to resolve the Golgi cisternae and the ER-Golgi interface. The data presented here shows that Atgolgin-84A seems to be localised at a pre-cis-Golgi compartment that is also labelled by one of the COPII proteins. Optical trapping is a technology in which an infrared laser beam can be used to capture and manipulate Golgi bodies in planta. The trapping experiments using optical tweezers revealed differences in Golgi bodies trapping properties when the truncated version Atgolgin-84A1-557 was overexpressed in N. tabacum leaves. Under the hypothesis that Atgolgin-84A could also be implicated in ER to Golgi trafficking a secretion assay was optimised using Atgolgin-84A as effector expected to affect the transport of cargo molecules. The trafficking of cargo was imaged when cells were expressing Atgolgin-84A full-length and mutant Atgolgin-84A without coiled-coil domains. The cargo was redirected to a different compartment. This hypothesis was also tested using an α-amylase assay and the secretion index decreased when Atgolgin-84A was co-expressed with cargo supporting the effect observed using confocal imaging. The results show strong evidence for a role of Atgolgin-84A in trafficking between ER and Golgi.

Grantor dc:publisher

Oxford Brookes University

Year dc:date

2018

Author dc:creator

• Vieira, Vanessa

Contributors dc:contributor

• Hawes, Chris
• Kriechbaumer, Verena

Statement dc:rights

• All rights reserved

Language dc:language

en

DOI dc:identifier

https://doi.org/10.24384/ec9c-rd45

OAI identifier oai:identifier

tle:5459f244-c872-4c0c-993c-13dcdfaa823d:d6bd9758-527a-46cd-bfe2-c433766e8fca:1