In situ artificial contact sites (ISACS) between synthetic and endogenous organelle membranes allow for quantification of protein-tethering activities.

ABSTRACT

Membrane contact sites are specialized areas where the membranes of two distinct organelles are physically connected and allow for the exchange of molecules and for signaling processes. Understanding the mechanisms whereby proteins localize to and function in these structures is of special interest; however, methods allowing for reconstitution of these contact sites are few and only based on synthetic membranes and recombinant proteins. Here, we devised a strategy to create in situ artificial contact sites between synthetic and endogenous organelle membranes. Liposomes functionalized with a peptide containing a two phenylalanines in an acidic tract (FFAT) motif were added to adherent cells whose plasma membrane was perforated. Confocal and super-resolution microscopy revealed that these liposomes associated with the endoplasmic reticulum via the specific interaction of the FFAT motif with endoplasmic reticulum-resident vesicle-associated membrane protein-associated proteins. This approach allowed for quantification of the attachment properties of peptides corresponding to FFAT motifs derived from distinct proteins and of a protein construct derived from steroidogenic acute regulatory protein-related lipid transfer domain-3. Collectively, these data indicate that the creation of in situ artificial contact sites represents an efficient approach for studying the membrane-tethering activity of proteins and for designing membrane contact site reconstitution assays in cellular contexts.