Christensen, N. R. | bioRxiv Science

bioRxiv2021

Bi-directional protein-protein interactions control liquid-liquid phase separation of PSD-95 and its interaction partners

The organization of the postsynaptic density (PSD), a protein-dense semi-membraneless organelle, is mediated by numerous specific protein-protein interactions (PPIs) which constitute a functional post-synapse. Postsynaptic density protein 95 (PSD-95) interacts with a manifold of proteins, including the C-terminal of transmembrane AMPA receptor (AMAPR) regulatory proteins (TARPs). Here, we uncover the minimal essential peptide responsible for the stargazin (TARP-{gamma}2) mediated liquid-liquid phase separation (LLPS) formation of PSD-95 and other key protein constituents of the PSD. Furthermore, we find that pharmacological inhibitors of PSD-95 can facilitate formation of LLPS. We found that in some cases LLPS formation is dependent on multivalent interactions while in other cases short peptides carrying a high charge are sufficient to promote LLPS in complex systems. This study offers a new perspective on PSD-95 interactions and their role in LLPS formation, while also considering the role of affinity over multivalency in LLPS systems.

biochemistry↗

bioRxiv2020

Coding variants identified in diabetic patients alter PICK1 BAR domain function in insulin granule biogenesis

Bin/amphiphysin/Rvs (BAR) domains are positively charged crescent-shaped modules that shape negatively charged curved lipid membranes during membrane remodeling processes. The BAR domain proteins ICA69, PICK1 and arfaptins have recently been demonstrated to coordinate the budding and formation of immature secretory granules (ISGs) at the trans-Golgi network. Here, we identify four coding variants in the PICK1 gene from a Danish whole-exome screening of diabetic patients, that all involve change of positively charged residues in the PICK1 BAR domain. All four coding variants failed to rescue the insulin content in INS-1E cells upon KD of endogenous PICK1. Moreover, two variants showed dominant negative properties. Interestingly, in vitro assays addressing the BAR domain function suggest that the coding variants compromised membrane binding capacity but increased capacity to cause fission of liposomes. Live confocal microscopy and super-resolution microscopy further revealed that PICK1 resides transiently on ISGs before egress via vesicular budding events. Interestingly, this egress of PICK1 was accelerated in the coding variants. We propose that PICK1 assists or complements the removal of excess membrane and generic membrane trafficking proteins, and possibly also insulin from ISGs during the maturation process and that the coding variants may cause premature budding possibly explaining their dominant negative function.

cell biology↗