The potential role of liquid-liquid phase separation in the cellular fate of the compartments for unconventional protein secretion.

Eukaryotic cells have developed intricate mechanisms for biomolecule transport, particularly in stressful conditions. This interdisciplinary study delves into unconventional protein secretion (UPS) pathways activated during starvation, facilitating the export of proteins bypassing most of the components of the classical secretory machinery. Specifically, we focus on the underexplored mechanisms of the GRASP's role in UPS, particularly in biogenesis and cargo recruitment for the vesicular-like compartment for UPS. Our results show that liquid-liquid phase separation (LLPS) plays a key role in the coacervation of Grh1, the GRASP yeast homologue, under starvation-like conditions. This association seems a precursor to the Compartment for Unconventional Protein Secretion (CUPS) biogenesis. Grh1's self-association is regulated by electrostatic, hydrophobic, and hydrogen-bonding interactions. Importantly, our study demonstrates that phase-separated states of Grh1 can recruit UPS cargo under starvation-like situations. Additionally, we explore how the coacervate liquid-to-solid transition could impact cells' ability to return to normal post-stress states. Our findings offer insights into intracellular protein dynamics and cell adaptive responses to stress.

Exploring liquid-liquid phase separation in the organisation of Golgi matrix proteins.

The Golgi apparatus is a critical organelle in protein sorting and lipid metabolism. Characterized by its stacked, flattened cisternal structure, the Golgi exhibits distinct polarity with its cis- and trans-faces orchestrating various protein maturation and transport processes. At the heart of its structural integrity and organisation are the Golgi Matrix Proteins (GMPs), predominantly comprising Golgins and GRASPs. These proteins contribute to this organelle's unique stacked and polarized structure and ensure the precise localization of Golgi-resident enzymes, which is crucial for accurate protein processing. Despite over a century of research since its discovery, the Golgi architecture's intricate mechanisms still need to be fully understood. Here, we discuss that GMPs across different Eukaryotic lineages present a significant tendency to form biomolecular condensates. Moreover, we validated experimentally that members of the GRASP family also exhibit a strong tendency. Our findings offer a new perspective on the possible roles of protein disorder and condensation of GMPs in the Golgi organisation.

A gold revision of the Golgi Dynamics (GOLD) domain structure and associated cell functionalities.

The classical secretory pathway is the key membrane-based delivery system in eukaryotic cells. Several families of proteins involved in the secretory pathway, with functionalities going from cargo sorting receptors to the maintenance and dynamics of secretory organelles, share soluble globular domains predicted to mediate protein-protein interactions. One of them is the 'Golgi Dynamics' (GOLD) domain, named after its strong association with the Golgi apparatus. There are many GOLD-containing protein families, such as the transmembrane emp24 domain-containing proteins (TMED/p24 family), animal SEC14-like proteins, human Golgi resident protein ACBD3, a splice variant of TICAM2 called TRAM with GOLD domain, and FYCO1. Here, we critically review the state-of-the-art knowledge of the structures and functions of the main representatives of GOLD-containing proteins in vertebrates. We provide the first unified description of the GOLD domain structure across different families since the first high-resolution structure was determined. With a brand-new update on the definition of the GOLD domain, we also discuss how its tertiary structure fits the ß-sandwich-like fold map and give exciting new directions for forthcoming studies.

Conformational flexibility of GRASPs and their constituent PDZ subdomains reveals structural basis of their promiscuous interactome.

The Golgi complex is a central component of the secretory pathway, responsible for several critical cellular functions in eukaryotes. The complex is organized by the Golgi matrix that includes the Golgi reassembly and stacking protein (GRASP), which was shown to be involved in cisternae stacking and lateral linkage in metazoan. GRASPs also have critical roles in other processes, with an unusual ability to interact with several different binding partners. The conserved N terminus of the GRASP family includes two PSD-95, DLG, and ZO-1 (PDZ) domains. Previous crystallographic studies of orthologues suggest that PDZ1 and PDZ2 have similar conformations and secondary structure content. However, PDZ1 alone mediates nearly all interactions between GRASPs and their partners. In this work, NMR, synchrotron radiation CD, and molecular dynamics (MD) were used to examine the structure, flexibility, and stability of the two constituent PDZ domains. GRASP PDZs are structured in an unusual ß3 α1 ß4 ß5 α2 ß6 ß1 ß2 secondary structural arrangement and NMR data indicate that the PDZ1 binding pocket is formed by a stable ß2 -strand and a more flexible and unstable α2 -helix, suggesting an explanation for the higher PDZ1 promiscuity. The conformational free energy profiles of the two PDZ domains were calculated using MD simulations. The data suggest that, after binding, the protein partner significantly reduces the conformational space that GRASPs can access by stabilizing one particular conformation, in a partner-dependent fashion. The structural flexibility of PDZ1, modulated by PDZ2, and the coupled, coordinated movement between the two PDZs enable GRASPs to interact with multiple partners, allowing them to function as promiscuous, multitasking proteins.