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Protein Sorting in the Endomembrane System

• Juan S. Bonifacino, PhD, Head, Section on Intracellular Protein Trafficking
• Rafael Mattera, PhD, Staff Scientist
• Xiaolin Zhu, Nurse, Technician
• Nireekshit Addanki Tirumala, PhD, Visiting Fellow
• Raffaella De Pace, PhD, Visiting Fellow
• Saikat Ghosh, PhD, Visiting Fellow
• Morié Ishida, PhD, Visiting Fellow
• Ganesh Shelke, PhD, Visiting Fellow
• Adriana Golding, PhD, Intramural Research Training Award Fellow
• Jennifer M. Kunselman, PhD, Intramural Research Training Award Fellow
• Nicole Hsiao-Sánchez, BS, Postbaccalaureate Fellow
• Bob Hsieh, BS, Postbaccalaureate Fellow
• Robert Kaltenbach, BS, Postbaccalaureate Fellow

Our laboratory studies the molecular mechanisms underlying the sorting of transmembrane proteins (known as cargo) to various compartments within the endomembrane system of eukaryotic cells. The system comprises an array of membrane-enclosed organelles including the endoplasmic reticulum (ER), the Golgi apparatus, the trans-Golgi network (TGN), endosomes, lysosomes, lysosome-related organelles (LROs, e.g., melanosomes, cytotoxic granules), and various domains of the plasma membrane in polarized cells such as epithelial cells and neurons. The transport of cargo between these compartments is mediated by vesicular or tubular carriers that bud from a donor compartment, translocate through the cytoplasm, and fuse with an acceptor compartment. We study the molecular machineries that mediate these processes in the context of different intracellular transport pathways, including endocytosis, recycling from endosomes to the plasma membrane, retrograde transport from endosomes to the TGN, biogenesis of lysosomes and LROs, autophagy, and polarized sorting in epithelial cells and neurons. Our fundamental research serves as a basis for explaining the pathogenetic mechanisms of protein traffic disorders, including the pigmentation and bleeding disorder Hermansky-Pudlak syndrome (HPS), hereditary spastic paraplegias (HSPs), and other neuro-developmental and early infantile neuro-degenerative disorders.

The adaptor protein chaperone AAGAB promotes assembly of the AP-4 complex.

Adaptor protein 4 (AP-4) is a heterotetrameric complex, composed of epsilon, beta4, mu4, and sigma4 subunits, that mediates export of transmembrane cargos, including autophagy protein 9A (ATG9A), from the TGN towards pre-autophagosomal structures (Figure 1). AP-4 has received particular attention in recent years because mutations in any of its subunits cause a complicated form of hereditary spastic paraplegia referred to as “AP-4–deficiency syndrome.” This year, we reported that the alpha- and gamma-adaptin–binding protein (AAGAB, also known as p34) binds to and stabilizes AP-4 subunits, thus promoting complex assembly. The physiological importance of these interactions is underscored by the observation that AAGAB–knockout (KO) cells exhibit reduced levels of AP-4 subunits and accumulation of ATG9A at the TGN, like those in cells with mutations in AP-4–subunit genes. These findings demonstrated that AP-4 assembly is not spontaneous but AAGAB–assisted, further contributing to the understanding of an adaptor protein complex that is critically involved in the development of the central nervous system.

Contribution to the development of intrathecal AAV9/AP4M1 gene therapy for hereditary spastic paraplegia 50 caused by mutations in the mu4 subunit of AP-4

We also contributed to the development of gene therapy for hereditary spastic paraplegia 50 (SPG50) in collaboration with Xin Chen, Steven Gray, and other colleagues. SPG50 is a rare childhood-onset neurological disorder caused by mutations in the AP4M1 gene. Our laboratory demonstrated that infection of skin fibroblasts from patients having AP4M1 mutations with an AAV2–AP4M1 vector rescued the assembly of the AP-4 complex and the export of ATG9A from the trans-Golgi network. Our collaborators then showed that intrathecal injection of a similar AAV9–AP4M1 vector had an acceptable safety profile in mice, rats, and non-human primates, and resulted in partial correction of phenotypic defects in AP4M1–KO mice, preclinical results that support an investigational gene transfer clinical trial to treat SPG50.

Architecture of the ESCPE-1 membrane coat: unveiling a key process of endosomal sorting

Intracellular recycling of membrane proteins plays a crucial role in re-using receptors, ion channels, and transporters. A critical player in this recycling machinery is the endosomal sorting complex for promoting exit 1 (ESCPE-1), responsible for rescuing transmembrane proteins from the endolysosomal pathway and transporting them to the trans-Golgi network and the plasma membrane. We collaborated with the laboratories of Aitor Hierro and Daniel Castaño-Díez to conduct biochemical, structural, and functional analyses into the organization of ESCPE-1, revealing that the complex forms a coat with a single-layer architecture. Furthermore, we found that synergistic interactions between ESCPE-1 protomers, phosphoinositides, and cargo molecules lead to the global arrangement of amphipathic helices, driving the formation of recycling tubules.

Endolysosome fusion attenuates exosome secretion.

In previous research, we discovered an eight-subunit complex named BORC, which plays a crucial role in recruiting the small GTPase ARL8, kinesin motor proteins, and the tethering factor HOPS to late endosomes and lysosomes. This past year, we reported that the BORC–ARL8–HOPS axis is responsible for regulating exosome secretion. Exosomes are small vesicles that cells release to dispose of undegraded materials and facilitate intercellular communication. A major source of exosomes is intraluminal vesicles within multi-vesicular endosomes, which can undergo either exocytic fusion with the plasma membrane or fusion with lysosomes. The factors that determine these alternative fates, however, were previously unknown. Our findings showed that disrupting the BORC–ARL8–HOPS axis impairs endolysosomal fusion, preventing the delivery of intraluminal vesicles to lysosomes and thus increasing exosome secretion. Our findings additionally suggested that targeting the BORC–ARL8–HOPS pathway may be a promising strategy to enhance exosome yields for biotechnology applications.

Small GTPases coordinate HOPS–mediated tethering of late endosomes and lysosomes

The transportation of endocytosed cargoes to lysosomes relies on HOPS–dependent tethering of late endosomes to lysosomes prior to fusion. Although several proteins interact with HOPS, their exact localization and involvement in the tethering process remained unclear. To address this problem, we collaborated with Albert Haas and Andreas Jeschke to determine the order and functional interdependence of HOPS and its interacting proteins in cargo transport to lysosomes. Our findings revealed that the small GTPases RAB2A and RAB7 are associated with late endosomes, while the small GTPase ARL8 and the BORC complex localize to lysosomes. HOPS facilitates late endosome-lysosome fusion by bridging late endosomal RAB2A with lysosomal BORC–anchored ARL8. Additionally, we observed that RAB7 is not present at HOPS–dependent tethering sites, but promotes fusion by facilitating the movement of late endosomes via dynein.

ARF1-related disorder: unveiling pathophysiological mechanisms

ADP–ribosylation factor 1 (ARF1) is a small GTPase that plays a critical role in regulating membrane traffic at the Golgi apparatus and endosomes by interacting with various coat proteins and lipid-modifying enzymes. In collaboration with the laboratory of Tyler Pierson, we reported this past year a pediatric patient with an ARF1–related disorder caused by a monoallelic de novo missense variant (c.296 G > A; p.R99H) in the ARF1 gene. The patient presented with developmental delay, hypotonia, intellectual disability, and motor stereotypies. Our functional analysis of the R99H–ARF1 variant protein showed that it was expressed at normal levels and correctly localized to the Golgi apparatus. However, the expression of this variant led to swelling of the Golgi apparatus and increased recruitment of coat proteins, as well as altered the morphology of recycling endosomes. Furthermore, protein interaction analyses indicated that R99H–ARF1 exhibited a stronger binding affinity to the ARF1–effector GGA3 than to wild-type ARF1, properties that suggested that the pathogenetic mechanism of the R99H–ARF1 variant involves constitutive activation, leading to Golgi and endosomal alterations. The study contributed to our understanding of the genetic basis of neuro-developmental disorders associated with ARF1 variants, shedding light on the pathophysiological mechanisms underlying these conditions.

Publications

1. Bonifacino JS. Getting where you want to go. Mol Biol Cell 2022 33:ae4.
2. Mattera R, De Pace R, Bonifacino JS. The adaptor protein chaperone AAGAB stabilizes AP-4 complex subunits. Mol Biol Cell 2022 33:ar109.
3. Lopez-Robles C, Scaramuzza S, Astorga-Simon EN, Ishida M, Williamson CD, Baños-Mateos S, Gil-Carton D, Romero-Durana M, Vidaurrazaga A, Fernandez-Recio J, Rojas AL, Bonifacino JS, Castaño-Díez D, Hierro A. Architecture of the ESCPE-1 membrane coat. Nat Struct Mol Biol 2023 30:958–969.
4. Shelke GV, Williamson CD, Jarnik M, Bonifacino JS. Inhibition of endolysosome fusion increases exosome secretion. J Cell Biol 2023 222:e202209084.
5. Chen X, Dong T, Hu Y, De Pace R, Mattera R, Eberhardt K, Ziegler M, Pirovolakis T, Sahin M, Bonifacino JS, Ebrahimi-Fakhari D, Gray SJ. Intrathecal AAV9/AP4M1 gene therapy for hereditary spastic paraplegia 50 shows safety and efficacy in preclinical studies. J Clin Invest 2023 133:e164575.
6. Ishida M, Otero MG, Freeman C, Sánchez-Lara PA, Guardia CM, Pierson TM, Bonifacino JS. A neurodevelopmental disorder associated with an activating de novo missense variant in ARF1. Hum Mol Genet 2023 32:1162–1174.
7. De Pace R, Bonifacino JS. Phagocytosis: Phagolysosome vesiculation promotes cell corpse degradation. Curr Biol 2023 33:R143–R146.
8. Schleinitz A, Pöttgen LA, Keren-Kaplan T, Pu J, Saftig P, Bonifacino JS, Haas A, Jeschke A. Consecutive functions of small GTPases guide HOPS-mediated tethering of late endosomes and lysosomes. Cell Rep 2023 42:111969.
9. Zhang Z, Venditti R, Ran L, Liu Z, Vivot K, Schürmann A, Bonifacino JS, De Matteis MA, Ricci R. Distinct changes in endosomal composition promote NLRP3 inflammasome activation. Nat Immunol 2023 24:30–41.
10. Jani RA, Di Cicco A, Keren-Kaplan T, Vale-Costa S, Hamaoui D, Hurbain I, Tsai FC, Di Marco M, Macé AS, Zhu Y, Amorim MJ, Bassereau P, Bonifacino JS, Subtil A, Marks MS, Lévy D, Raposo G, Delevoye C. PI4P and BLOC-1 remodel endosomal membranes into tubules. J Cell Biol 2022 221:e202110132.

Collaborators

• Xin Chen, PhD, University of Texas Southwestern Medical Center, Dallas, TX
• Cédric Delevoye, PhD, Institut Curie, INSERM, Paris, France
• Antonella De Matteis, MD, TIGEM, Pozzuoli, Italy
• Steven Gray, PhD, University of Texas Southwestern Medical Center, Dallas, TX
• Albert Haas, PhD, University of Bonn, Bonn, Germany
• Aitor Hierro, PhD, CIC bioGUNE, Bilbao, Spain
• Andreas Jeschke, PhD, University of Bonn, Bonn, Germany
• Daniel Lévy, PhD, Institut Curie, INSERM, Paris, France
• Tyler Pierson, MD, PhD, Cedars-Sinai, Los Angeles, CA
• Graça Raposo, PhD, Institut Curie, INSERM, Paris, France
• Romeo Ricci, MD, University of Strasbourg, Strasbourg, France

Contact

For more information, email juan.bonifacino@nih.gov or visit https://www.nichd.nih.gov/research/atNICHD/Investigators/bonifacino.

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