312 Proc. Jpn. Acad., Ser. B 95 (2019) [Vol. 95, Review Roles of protrudin at interorganelle membrane contact sites † By Michiko SHIRANE*1, (Communicated by Takao SEKIYA, M.J.A.) Abstract: Intracellular organelles were long viewed as isolated compartments floating in the cytosol. However, this view has been radically changed within the last decade by the discovery that most organelles communicate with the endoplasmic reticulum (ER) network via membrane contact sites (MCSs) that are essential for intracellular homeostasis. Protrudin is an ER resident protein that was originally shown to regulate neurite formation by promoting endosome trafficking. More recently, however, protrudin has been found to serve as a tethering factor at MCSs. The roles performed by protrudin at MCSs are mediated by its various domains, including inactivation of the small GTPase Rab11, bending of the ER membrane, and functional interactions with other molecules such as the motor protein KIF5 and the ER protein VAP. Mutations in the protrudin gene (ZFYVE27) are associated with hereditary spastic paraplegia, an axonopathy that results from defective ER structure. This review, examines the pleiotropic molecular functions of protrudin and its role in interorganellar communication. Keywords: protrudin, ZFYVE27, endoplasmic reticulum, membrane contact sites, organelle, trafficking network via membrane contact sites (MCSs) Introduction throughout the cell. MCSs are microdomains where The focus of research on intracellular organelles the membranes of the ER and other organelles come of eukaryotic cells has shifted dramatically in recent into close proximity (within a distance of <30 nm) in years from the characterization of each organelle order to communicate with each other. However, the compartment separately to the study of intercom- two membranes do not fuse, but rather maintain partment communication. It is now widely accepted their separate identities.1)–4) MCSs were first identi- that organelles do not function independently but fied decades ago by classical electron microscopy, but rather interact with the endoplasmic reticulum (ER) the mechanisms underlying their formation and function remained unclear until recently. The devel- *1 Department of Molecular Biology, Graduate School of opment of advanced imaging technologies, such as Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi, Japan. three-dimensional, time-lapse, and high-resolution † Correspondence should be addressed: M. Shirane, Depart- microscopy, as well as of fluorescent proteins for the ment of Molecular Biology, Graduate School of Pharmaceutical visualization of MCSs, have made possible the recent Sciences, Nagoya City University, 3-1 Tanabe-Dori, Mizuho-ku, progress in this area of research. MCSs have been Nagoya, Aichi 467-8603, Japan (e-mail: [email protected] cu.ac.jp). shown to mediate lipid transfer, calcium ion regu- Abbreviations: CC: coiled-coil; ER: endoplasmic reticu- lation, and organelle dynamics such as endosome lum; ERAD: endoplasmic reticulum-associated degradation; ERK: trafficking and mitochondrial fission (Fig. 1).5) Teth- extracellular signal-regulated kinase; FFAT: two phenylalanines in ering factors that link the apposing membranes at an acidic tract; FKBP38: FK506 binding protein 38; FYVE: Fab1, YOTB, Vac1, EEA1; GDI: guanine nucleotide dissociation MCSs have also recently been discovered and shown inhibitor; HP: hairpin; HSP: hereditary spastic paraplegia; LCR: to play important roles in the formation and function low complexity region; MCS: membrane contact site; MSP: major of these structures. sperm protein; NGF: nerve growth factor; PIPs: phosphatidylino- Many organelles communicate with the ER via sitol phosphate; RBD11: Rab11 binding domain; SPG: spastic paraplegia; TM: transmembrane; VAP: vesicle-associated mem- MCSs. The ER is a continuous membrane system brane protein-associated protein. that comprises the nuclear envelope, ribosome- doi: 10.2183/pjab.95.023 ©2019 The Japan Academy No. 7] Roles of protrudin at membrane contact sites 313 HeLa COS7 MCS Endosome < 30 nm ER FLAG-protrudin Tethering factor ・Lipid transfer ・Ca2+ regulation ・Organelle dynamics Fig. 1. (Color online) Structure and functions of membrane contact sites (MCSs). MCSs are microdomains that form Mock between the ER and other organelles by juxtaposition of the organelle membranes. The apposing membranes approach each other to within a distance of <30 nm in order to support communication, but they do not fuse and, therefore, retain their Fig. 2. (Color online) Protrudin promotes process formation. separate identities. Well-established functions of MCSs include Overexpression of protrudin was found to promote process mediation of lipid transfer, calcium ion regulation, and organelle formation in nonneuronal cells such as HeLa and COS7 cells. dynamics including endosome trafficking and mitochondrial The upper panels show immunofluorescence staining of FLAG fission. Tethering factors at MCSs play key roles in the function epitope-tagged protrudin in the transfected cells, and the lower of these structures. panels are phase-contrast micrographs of mock-transfected cells. studded peripheral sheets (rough ER), and an interconnected tubular network (smooth ER). The ity and the subsequent formation of long protrusions structure of the smooth ER is supported by a group similar to neurites when it was overexpressed in of membrane curvature proteins that contain a cultured cells (Fig. 2). Thus, we named this protein hydrophobic hairpin domain. Impaired function of “protrudin” on the basis of this activity.6) Since its these proteins results in the development of heredi- discovery, we and others have uncovered molecular tary spastic paraplegia (HSP), a neurological disease mechanisms underlying this process formation and caused by abnormal ER structure and function and other physiological functions of protrudin (Fig. 3). the consequent evocation of ER stress and axonal Analysis of the predicted amino acid sequence degeneration. of protrudin for functional domains revealed a Rab11 Protrudin is a protein that plays a central role binding domain (RBD11), two transmembrane (TM) in directional endosomal trafficking by functioning as domains, a hairpin (HP) domain, a low complexity a tethering factor at MCSs.6),7) At MCSs formed region (LCR), an two phenylalanines in an acidic between the ER and endosomes, protrudin promotes tract (FFAT) motif, a coiled-coil domain, and a the transfer of endosomes from the ER to micro- Fab1, YOTB, Vac1, EEA1 (FYVE) domain (Fig. 4). tubules for their polarized transport. In addition, These structural characteristics provided clues to the protrudin contributes to the regulation of ER functions of protrudin in the regulation of organelle structure. Mutations in the protrudin gene are also dynamics such as directional endosome trafficking associated with HSP, suggesting that the integrity of and ER morphogenesis. endosome trafficking and ER structure regulated by protrudin is essential for the maintenance of neurons Protrudin regulates Rab11-dependent ffi that extend long axons. polarized tra cking Certain types of cell, such as epithelial cells and Discovery and structural characteristics neurons, are polarized and possess distinct plasma of protrudin membrane domains. Epithelial cells thus manifest Protrudin was first discovered as a protein of apical and basolateral domains that face a lumen, unknown function that binds to FK506 binding such as the airway in the lungs, and the interior of the protein 38 (FKBP38), a multifunctional membrane body, respectively. Similarly, neurons possess axonal chaperone that localizes to mitochondria and the and somatodendritic domains, which are specialized ER.8)–11) In a yeast two-hybrid screen to select for the transmission of signals to and the reception proteins that bind to FKBP38, we identified a novel of those from other cells, respectively. Cell polarity is protein that induced pronounced membrane deform- largely dependent on recycling endosome traffic, or 314 M. SHIRANE [Vol. 95, Protrudin promotes neurite Protrudin interacts with Discovery of neuron-specific Defects in ER structure causes formation via Rab11 VAP at the ER splicing of protrudin mRNA HSP due to protrudin mutation (Ref. 6) (Ref. 18) (Ref. 24) (Ref. 12) Hyperphosphorylation of Protrudin promotes polarized Generation of SRRM4 regulates Discovery of protrudin in FKBP38-KO mice trafficking with KIF5 protrudin-KO mice splicing of protrudin mRNA protrudin (Ref. 8, 9) (Ref. 19) (Ref. 24) (Ref. 25) 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 Mutation of protrudin gene Protrudin-FYVE binds Protrudin regulates Protrudin regulates endosome in HSP patient family to PIPs ER structure trafficking via MCSs (Ref. 13) (Ref. 23) (Ref. 17) (Ref. 7) Fig. 3. (Color online) History of protrudin research. Major discoveries relating to protrudin are summarized. KO, knockout. LCR RBD11/GDI HP FFAT CC TM L FYVE Rab11 ER Rab7 VAP KIF5 PIPs GDP GTP Membrane tethering Lyso-endosome Polarized trafficking Microtubule-dependent transport endosome trafficking ER structure MCSs Fig. 4. (Color online) Domain structure of protrudin. Protrudin contains multiple functional domains. The Rab11 binding domain (RBD11) interacts with the GDP-bound form of Rab11 and thereby promotes polarized endosome trafficking. The amino acid sequence of protrudin RBD11 is similar to the corresponding sequence of guanine nucleotide dissociation inhibitors (GDIs). Protrudin resides at the ER, and its two