Subcellular localisation of an endoplasmic reticulum-plasma membrane tethering factor, SYNAPTOTAGMIN 1, is affected by fluorescent protein fusion

ABSTRACTMembrane contact sites (MCS) have increasingly received attention because of their general role in a number of important cellular processes. SYNAPTOTAGMIN 1 (SYT1) is a tethering factor connecting the endoplasmic reticulum (ER) and the plasma membrane (PM) in plant cells. Confocal microscopy using fluorescent protein fusion is an indispensable tool for studying protein localisation and functions. However, several studies have reported that fluorescent protein dimerisation affects the subcellular localisation of proteins tagged by the fluorescent protein. Here, we investigate the effects of fluorescent protein dimerisation by comparing the subcellular localisation of SYT1 fused with a synthetic GFP (SYT1-sGFP) and SYT1 fused with a monomeric GFP (SYT1-mGFP). SYT1-mGFP was confined to specific domains in the ER, whereas SYT1-sGFP spread along the ER when transiently overexpressed. SYT1-localised regions were suggested to correspond to ER-PM contact sites because of its immobility. Similar results we...