BACKGROUND: Cholesterol plays a vital role in multiple physiological processes. Cellular uptake of cholesterol is mediated primarily through endocytosis of low-density lipoprotein (LDL) receptor. New modifiers of this process remain to be characterized. Particularly, the role of fasting- and CREB-H-induced (FACI) protein in cholesterol homeostasis merits further investigation.
METHODS: Interactome profiling by proximity labeling and affinity purification - mass spectrometry was performed. Total internal reflection fluorescence microscopy and confocal immunofluorescence microscopy were used to analyze protein co-localization and interaction. Mutational analysis was carried out to define the domain and residues required for FACI localization and function. Endocytosis was traced by fluorescent cargos. LDL uptake in cultured cells and diet-induced hypercholesterolemia in mice were assessed.
RESULTS: FACI interacted with proteins critically involved in clathrin-mediated endocytosis, vesicle trafficking, and membrane cytoskeleton. FACI localized to clathrin-coated pits (CCP) on plasma membranes. FACI contains a conserved DxxxLI motif, which mediates its binding with the adaptor protein 2 (AP2) complex. Disruption of this motif of FACI abolished its CCP localization but didn\'t affect its association with plasma membrane. Cholesterol was found to facilitate FACI transport from plasma membrane to endocytic recycling compartment in a clathrin- and cytoskeleton-dependent manner. LDL endocytosis was enhanced in FACI-overexpressed AML12 cells but impaired in FACI-depleted HeLa cells. In vivo study indicated that hepatic FACI overexpression alleviated diet-induced hypercholesterolemia in mice.
CONCLUSIONS: FACI facilitates LDL endocytosis through its interaction with the AP2 complex.

Orchestration of a complex network of protein interactions drives clathrin-mediated endocytosis (CME). A central role for the AP2 adaptor complex beyond cargo recognition and clathrin recruitment has emerged in recent years. It is now apparent that AP2 serves as a pivotal hub for protein interactions to mediate clathrin coated pit maturation, and couples lattice formation to membrane deformation. As a key driver for clathrin assembly, AP2 complements the attenuating role of clathrin light chain subunits, which enable dynamic lattice rearrangement needed for budding. This review summarises recent insights into AP2 function with respect to CME dynamics and biophysics, and its relationship to the role of clathrin light chains in clathrin assembly.

The most represented components of clathrin-coated vesicles (CCVs) are clathrin triskelia and the adaptors clathrin assembly lymphoid myeloid leukemia protein (CALM) and the heterotetrameric complex AP2. Investigation of the dynamics of AP180-amino-terminal-homology (ANTH) recruitment during CCV formation has been hampered by CALM toxicity upon overexpression. We used knock-in gene editing to express a C-terminal-attached fluorescent version of CALM, while preserving its endogenous expression levels, and cutting-edge live-cell microscopy approaches to study CALM recruitment at forming CCVs. Our results demonstrate that CALM promotes vesicle completion upon membrane tension increase as a function of the amount of this adaptor present. Since the expression of adaptors, including CALM, differs among cells, our data support a model in which the efficiency of clathrin-mediated endocytosis is tissue specific and explain why CALM is essential during embryogenesis and red blood cell development.

Heterotetrameric clathrin adaptor protein complexes (APs) orchestrate the formation of coated vesicles for transport among organelles of the cell periphery. AP1 binds membranes enriched for phosphatidylinositol 4-phosphate, such as the trans Golgi network, while AP2 associates with phosphatidylinositol 4,5-bisphosphate of the plasma membrane. At their respective membranes, AP1 and AP2 bind the cytoplasmic tails of transmembrane protein cargo and clathrin triskelions, thereby coupling cargo recruitment to coat polymerization. Structural, biochemical and genetic studies have revealed that APs undergo conformational rearrangements and reversible phosphorylation to cycle between different activity states. While membrane, cargo and clathrin have been demonstrated to promote AP activation, growing evidence supports that membrane-associated proteins such as Arf1 and FCHo also stimulate this transition. APs may be returned to the inactive state via a regulated process involving phosphorylation and a protein called NECAP. Finally, because antiviral mechanisms often rely on appropriate trafficking of membrane proteins, viruses have evolved novel strategies to evade host defenses by influencing the conformation of APs. This review will cover recent advances in our understanding of the molecular inputs that stimulate AP1 and AP2 to adopt structurally and functionally distinct configurations.

Despite its biological and medical relevance, traffic from the Golgi to the plasma membrane (PM) is one of the least understood steps of secretion. Exomer is a protein complex that mediates the trafficking of certain cargoes from the trans-Golgi network/early endosomes to the PM in budding yeast. Here, we show that in Schizosaccharomyces pombe the Cfr1 and Bch1 proteins constitute the simplest form of an exomer. Cfr1 co-immunoprecipitates with Assembly Polypeptide adaptor 1 (AP-1), AP-2, and Golgi-localized, gamma-adaptin ear domain homology, ARF-binding (GGA) subunits, and cfr1+ interacts genetically with AP-1 and GGA genes. Exomer-defective cells exhibit multiple mild defects, including alterations in the morphology of Golgi stacks and the distribution of the synaptobrevin-like Syb1 protein, carboxypeptidase missorting, and stress sensitivity. S. pombe apm1Δ cells exhibit a defect in trafficking through the early endosomes that is severely aggravated in the absence of exomer. apm1Δ cfr1Δ cells exhibit a dramatic disorganization of intracellular compartments, including massive accumulation of electron-dense tubulovesicular structures. While the trans-Golgi network/early endosomes are severely disorganized in the apm1Δ cfr1Δ strain, gga21Δ gga22Δ cfr1Δ cells exhibit a significant disturbance of the prevacuolar/vacuolar compartments. Our findings show that exomer collaborates with clathrin adaptors in trafficking through diverse cellular compartments, and that this collaboration is important to maintain their integrity. These results indicate that the effect of eliminating exomer is more pervasive than that described to date, and suggest that exomer complexes might participate in diverse steps of vesicle transport in other organisms.

The Nef protein of the human immunodeficiency virus is a crucial determinant of viral pathogenesis and disease progression. Nef is abundantly expressed early in infection and is thought to optimize the cellular environment for viral replication. Nef controls expression levels of various cell surface molecules that play important roles in immunity and virus life cycle, by directly interfering with the itinerary of these proteins within the endocytic and late secretory pathways. To exert these functions, Nef physically interacts with host proteins that regulate protein trafficking. In recent years, considerable progress was made in identifying host-cell-interacting partners for Nef, and the molecular machinery used by Nef to interfere with protein trafficking has started to be unraveled. Here, we briefly review the knowledge gained and discuss new findings regarding the mechanisms by which Nef modifies the intracellular trafficking pathways to prevent antigen presentation, facilitate viral particle release and enhance the infectivity of HIV-1 virions.

The retinal pigment epithelium (RPE) comprises a monolayer of polarized pigmented epithelial cells that is strategically interposed between the neural retina and the fenestrated choroid capillaries. The RPE performs a variety of vectorial transport functions (water, ions, metabolites, nutrients and waste products) that regulate the composition of the subretinal space and support the functions of photoreceptors (PRs) and other cells in the neural retina. To this end, RPE cells display a polarized distribution of channels, transporters and receptors in their plasma membrane (PM) that is remarkably different from that found in conventional extra-ocular epithelia, e.g. intestine, kidney, and gall bladder. This characteristic PM protein polarity of RPE cells depends on the interplay of sorting signals in the RPE PM proteins and sorting mechanisms and biosynthetic/recycling trafficking routes in the RPE cell. Although considerable progress has been made in our understanding of the RPE trafficking machinery, most available data have been obtained from immortalized RPE cell lines that only partially maintain the RPE phenotype and by extrapolation of data obtained in the prototype Madin-Darby Canine Kidney (MDCK) cell line. The increasing availability of RPE cell cultures that more closely resemble the RPE in vivo together with the advent of advanced live imaging microscopy techniques provides a platform and an opportunity to rapidly expand our understanding of how polarized protein trafficking contributes to RPE PM polarity.