Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi


  • Mayuko Ichimura
  • Atsuko Nakanishi
  • Yasuko Kitagishi
  • Satoru Matsuda
Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101776


  RUFY1 : FYVE-finger containing protein; Rab4a interacting protein; Rab4aip; Rabip4; RUN and FYVE domain-containing 1; ZFYVE12

  RUFY2 : Denn; KIAA1537; LZ-FYVE; Rabip4R; RABIP4R; RUN and FYVE domain-containing 2; ZFYVE13

  RUFY3 : RIPX; Rap2 interacting protein X; RUN and FYVE domain-containing 3; SINGAR1; Single axon-related 1; ZFYVE30

  RUFY4 : RUN and FYVE domain-containing 4; ZFYVE31

Historical Background

The RUFY proteins (RUN and FYVE domain-containing protein family) have an amino-terminal RUN domain and a carboxyl-terminal FYVE domain, which associates with phosphatidylinositol 3-phosphate (PI3P) in early endosomes (Yang et al. 2002). As shown in Fig. 1, genome DNA analyses have revealed that a RUFY family consists of four members of RUFY proteins (Yoshida et al. 2010). The RUN domains, named from RPIP8, UNC-14, and NESCA proteins, might function as effectors of the small GTPase superfamily (MacDonald et al. 2004). For example, yeast two-hybrid assays for Rab binding activity of the RUN domains had shown the interaction of Rab33 with RUFY2/3. (Fukuda et al. 2011). Because many RUN domain-containing proteins are involved in the signaling of small GTPases in membranes, the RUN domain has been suggested to be involved in membrane trafficking (Yoshida et al. 2011; Kitagishi and Matsuda 2013). In particular, it has been suggested Rab family small GTPases and RUFY protein play a key role in the membrane trafficking (Mari et al. 2006; Kitagishi and Matsuda 2013). In addition, the RUN domain binds to some molecules including motor proteins, and the RUN domain might also be responsible for an interaction with a filamentous network. Actually, it has been revealed that RUFY proteins are localized predominantly to endosomes.
Rufy, Fig. 1

Schematic illustration indicating the domain structures of the RUFY1, RUFY2, RUFY3, and RUFY4 proteins. The functionally important sites and their interaction proteins are shown. Genomic locations of each human genes and approximate molecular mass of each the proteins are also shown at the ends. RUN RPIP8, UNC-14, and NESCA proteins; FYVE Fab-1, YGL023, Vps27, and EEA1 proteins; CC Coiled-Coil

Function of the RUN Domain on RUFY Family Proteins

The RUN domain consists of approximately 70 amino acids. Because RUN domains are frequently found in proteins involved in the regulation of Rab small GTPases, the RUN domain has been suggested to be involved in Rab-mediated cellular action. The RUN domain-containing proteins have been shown to promote endosomal fusion and are also significant for vesicular transport. In addition, the RUN domains appear to be required for localization to endosomal microdomains (Mari et al. 2001). Physical interaction between RUN proteins and filamentous materials has been confirmed by several biochemical experiments. Relationship among small GTPases, RUN proteins, and motor proteins may reflect a novel function for these proteins in the transport of vesicular cargoes within cells. For example, it has been reported that FYCO1 with a RUN domain works as an adapter linking autophagosomes to microtubule molecular motors and the Rab7, which is implicated in the phagosomal transport and fusion (Pankiv et al. 2010). A RUN domain of UNC-14 protein binds to the kinesin-1, a heterotetramer composed of kinesin heavy chain and kinesin light chain, and it regulates synaptic vesicle localization, which is predicted to play an important role in multiple Ras-like GTPase signaling pathways (Ogura and Goshima 2006). Because RUN domains are often found in proteins involved in the regulation of Rab family small GTPases, the RUN domain has been proposed to be involved in the Rab-mediated membrane trafficking. Accumulating data support the hypothesis that the RUN domain-containing proteins might be in charge of an interaction with a filamentous network linked to actin cytoskeleton and/or microtubules. There might be a collective role underlying the mechanism of the relationship of RUN domain to small GTPases and motor proteins. In addition, several downstream molecules of PI3K are involved in regulation of the membrane trafficking by networking with vesicle-associated RUN domain proteins such as RUFY proteins (Kitagishi and Matsuda 2013).


RUFY1, also known as RABIP4 (Rab4-interacting protein) or ZFYVE12 (Zinc finger FYVE domain-containing protein 12), is a 708 amino acid protein which localizes to the cytoplasm and/or the early endosomal membrane (Yang et al. 2002; He et al. 2009). RUFY1 has been identified as one of the downstream effectors of Etk protein kinase that is highly expressed in brain, lung, testis, and kidney. RUFY1 binds to PI3P-containing phospholipid vesicles and participates in early endosomal membrane trafficking (Yang et al. 2002). Downstream effects of PI3K signaling are facilitated by proteins containing a PI3P binding component designated by the FYVE finger domain. FYVE domain proteins are usually localized at the endosomes, then play an important role in endocytosis. Through the Src homology 2 (SH2) and Src homology 3 (SH3) domains of Etk, the Etk interacts with RUFY1, then, phosphorylates certain Tyr-residues of RUFY1, which is essential for the endosomal localization (Yang et al. 2002). The Etk plays an essential role in the regulation of endocytosis as a downstream effector of PI3K. Two coiled-coil domains also regulate endosomal localization of RUFY1 (Mari et al. 2001). Accordingly, the PI3K inhibitor wortmannin blocks the endosomal localization of RUFY1 (Mari et al. 2001). Rab14 engages in a GTP-dependent interaction with RUFY1 (Yamamoto et al. 2010). The active Rab14 controls RUFY1 recruitment onto endosomal membranes, and Rab4 allows the endosomal fusion (Yamamoto et al. 2010). The Rab14 seems to be a principal factor of RUFY1 recruitment to the endosomes, and the FYVE domain may assist RUFY1 targeting to PI3P-enriched early endosomes (Yamamoto et al. 2010). Both Rab14 and RUFY1 are involved in Rab4-dependent recycling endosome. Enlargement of early endosomes mediated by RUFY1 requires the interaction with Rab4. The RUFY1 is also present in the sorting endosomes, which would provide directional trafficking way from the recycling endosomes to the sorting endosomes. In addition, RUFY1 can modify the kinetic parameters of Glut1 protein recycling (Cormont et al. 2001). RUFY1 may have a role in regulating lysosome positioning through an interorganellar pathway (Ivan et al. 2012).


RUFY2, also known as RABIP4R (Rab4-interacting protein related) or ZFYVE13 (Zinc finger FYVE domain-containing protein 13), contains a RUN domain and a carboxyl terminal FYVE zinc finger, separated by two coiled-coil domains. RUFY2 is expressed in brain, lung, and testis localizing to the cellular nucleus. RUFY2 as well as RUFY1 interacts with the Etk that is a tyrosine kinase involved in regulation of various cellular processes. The carboxyl domain of RUFY2 binds to negative form of Rab33A. Transcriptomic data of a miRNA dosage-sensitive gene have revealed significant associations in a subset of genes such as RUFY2 (Bofill-De Ros et al. 2015). In addition, the RUFY2-RET in lung cancer has been identified as new therapeutically important gene fusions (Zheng et al. 2014). However, little is known about the precise intracellular functions as poorly characterized for RUFY2.


RUFY3, also known as RIPX (Rap2-interacting protein X) or SINGAR1 (single axon-related1), is an actin filament-related protein. RUFY3 is accumulated in growth cones and axons localized in hippocampal neurons, which has a role in the neuronal development. Appropriate expression of RUFY3 in neurons is required for the development of a single axon and axon growth (Fig. 2). RUFY3 specifically interacts with actin filament-binding proteins such as Fascin and colocalizes with Fascin in the neuronal growth cones. RUFY3 also contains the RUN domain and seems to play important roles in Ras-like small GTPase signaling pathway. In particular, Rab5 involves in a GTP-dependent interaction with RUFY3. RUFY3 can bind to the active Rab5 and weakly associates to Rap2 (Yoshida et al. 2010). So, RUFY3 may function as a docking protein for distinct two small GTPases. Neurons must generate a single axon and properly elongate the axon to reach its target for the accurate nervous system. Knockdown of RUFY3 impairs the distribution of actin filaments and Fascin, which results in shortened axons and increased proportion of neurons with multiple not single axons. RUFY3 certifies the robustness of neuronal polarity by suppressing formation of surplus axons (Mori et al. 2007). Therefore, RUFY3 may be important for precise neuronal axon elongation to control actin filament organization in neuronal growth cones. (Wei et al. 2014). Likewise, RUFY3 may confirm the strength of neuronal cell polarity by suppressing formation of extra axons.
Rufy, Fig. 2

Neuronal cells are asymmetric with discrete regions responsible for different roles. Arrows indicate the direction of polarity. Intracellular transport may be essential for the proper establishment of the cell polarity. RUFY3 and Rab5 in neurons may be required for the development of an axon and the axon growth. Note that several critical molecules have been omitted for clarity

Actin projection at the cell periphery is fundamental to the formation of invadopodia during cancer cell invasion and migration. RUFY3 encourages higher expression of several oncogenes. Besides, RUFY3 suppression inhibited anchorage-independent cell growth. Overexpression of RUFY3 leads to the formation of actin-enriched prominent structures at the cell periphery, which induces cancer cell migration. Furthermore, RUFY3 expression is correlated with tumor progression. It has been found significant upregulation of RUFY3 in cancer samples with highly invasive carcinoma. P21-activated kinase-1 (PAK1) promotes RUFY3 expression and interacts with RUFY3 (Fig. 3). In contrast, inhibition of PAK1 reduces RUFY3-induced cell migration and invasion. RUFY3 signaling could become a potential therapeutic strategy for cancer treatment (Wang et al. 2015). In addition, it has been reported that oxidized LDL-containing immune complexes affect the gene expression of RUFY3 in human U937 monocytic leukemia cells.
Rufy, Fig. 3

Roles of RUFY3 in cancer cells. RUFY3 and p21-activated kinase-1 (PAK1) may induce cancer cell progression, invasion, and metastasis


RUFY4 is a 571 amino acid protein that also contains a RUN domain and a FYVE zinc finger domain. The RUFY4 gene is located on chromosome 2 in human. RUFY4 is expressed in subsets of immune cells including dendritic cells (Fig. 4). RUFY4 may be a positive regulator of autophagy of the immune cells under specific conditions (Terawaki et al. 2016). Higher autophagy increases endogenous antigen presentation in RUFY4-expressing immune cells. In addition, RUFY4 impacts endosome dynamics in a subset of immune cells (Terawaki et al. 2015). RUFY4 expression leads to the reorganization of late endosomal compartments, thus varying the total endosome membrane dynamic as one of the Rab7 effectors during dendritic cells differentiation. RUFY4 overexpression increases the autophagic flux by stimulating both phagosome generation and supporting binding with lysosomes. Together with the Rab7, RUFY4 might be a regulatory element required for the autophagy correction in dendritic cells. Association of RUFY4 with Rab7 and PI3P-containing organelles is very similar to what is observed with Rubicon that leads to atypical morphology of late endosomes. Rubicon mediates the interaction between Beclin 1 and Rab7, which negatively regulates autophagosome and late endosome fusion “(Tabata et al. 2010)”. RUFY4 promotes the clustering and binding of lysosomes, which could facilitate fusion with autophagosomes. The other role of RUFY4 is supposed to be involved with zinc ion binding. RUFY4 seems to compete Rab7 for the same effector with Rubicon in order to control the appropriate autophagy.
Rufy, Fig. 4

Schematic illustration of dendritic cell-derived intracellular vesicle transport and signals. Following exposure to foreign antigen (Ag) by dendritic cells, the naive T cells are directed towards distinct developmental programs. RUFY4 and Rab7 may influence the endosomal dynamics in a subset of immune cells including the dendritic cell. Note that some critical trafficking routes have been omitted for clarity. Th T helper cells


RUN domain-containing proteins might be responsible for an interaction with a filamentous network linked to actin cytoskeleton and/or microtubules. In addition, several downstream molecules of PI3K are involved in the regulation of membrane trafficking by interacting with vesicle-associated RUFY family proteins, which may be activated in some of the cytoskeletal elements. The localization of RUFY proteins during membrane trafficking seems to be extremely dynamic. RUFY family consists of four members of RUFY proteins. RUFY1 binds to PI3P-containing phospholipid vesicles and participates in early endosomal membrane trafficking. RUFY2 as well as RUFY1 interacts with the Etk that is a tyrosine kinase involved in regulation of various cellular processes. RUFY3 may ensure the strength of neuronal cell polarity by suppressing formation of surplus axons. Furthermore, RUFY3 expression is correlated with tumor progression. RUFY4 may be a positive regulator of autophagy under specific immunological conditions. Interpreting the precise mechanisms involved in these processes will provide new insight into the physiological roles of the interesting proteins in regulating cellular functions. It is anticipated that future studies would address to get a better knowledge of the potential partners and novel roles of the RUFY proteins.


  1. Bofill-De Ros X, Santos M, Vila-Casadesús M, Villanueva E, Andreu N, Dierssen M, Fillat C. Genome-wide miR-155 and miR-802 target gene identification in the hippocampus of Ts65Dn Down syndrome mouse model by miRNA sponges. BMC Genomics. 2015;16:907.PubMedPubMedCentralCrossRefGoogle Scholar
  2. Cormont M, Mari M, Galmiche A, Hofman P, Le Marchand-Brustel Y. A FYVE-finger-containing protein, Rabip4, is a Rab4 effector involved in early endosomal traffic. Proc Natl Acad Sci USA. 2001;98:1637–42.PubMedPubMedCentralCrossRefGoogle Scholar
  3. Fukuda M, Kobayashi H, Ishibashi K, Ohbayashi N. Genome-wide investigation of the Rab binding activity of RUN domains: development of a novel tool that specifically traps GTP-Rab35. Cell Struct Funct. 2011;36:155–70.PubMedCrossRefGoogle Scholar
  4. He J, Vora M, Haney RM, Filonov GS, Musselman CA, Burd CG, Kutateladze AG, Verkhusha VV, Stahelin RV, Kutateladze TG. Membrane insertion of the FYVE domain is modulated by pH. Proteins. 2009;76:852–60.PubMedPubMedCentralCrossRefGoogle Scholar
  5. Ivan V, Martinez-Sanchez E, Sima LE, Oorschot V, Klumperman J, Petrescu SM, van der Sluijs P. AP-3 and Rabip4′ coordinately regulate spatial distribution of lysosomes. PLoS One. 2012;7:e48142.PubMedPubMedCentralCrossRefGoogle Scholar
  6. Kitagishi Y, Matsuda S. RUFY, Rab and Rap family proteins involved in a regulation of cell polarity and membrane trafficking. Int J Mol Sci. 2013;14:6487–98.PubMedPubMedCentralCrossRefGoogle Scholar
  7. MacDonald JI, Kubu CJ, Meakin SO. Nesca, a novel adapter, translocates to the nuclear envelope and regulates neurotrophin-induced neurite outgrowth. J Cell Biol. 2004;164:851–62.PubMedPubMedCentralCrossRefGoogle Scholar
  8. Mari M, Macia E, Le Marchand-Brustel Y, Cormont M. Role of the FYVE finger and the RUN domain for the subcellular localization of Rabip4. J Biol Chem. 2001;276:42501–8.PubMedCrossRefGoogle Scholar
  9. Mari M, Monzo P, Kaddai V, Keslair F, Gonzalez T, Le Marchand-Brustel Y, Cormont M. The Rab4 effector Rabip4 plays a role in the endocytotic trafficking of Glut 4 in 3 T3-L1 adipocytes. J Cell Sci. 2006;119:1297–306.PubMedCrossRefGoogle Scholar
  10. Mori T, Wada T, Suzuki T, Kubota Y, Inagaki N. Singar1, a novel RUN domain-containing protein, suppresses formation of surplus axons for neuronal polarity. J Biol Chem. 2007;282:19884–93.PubMedCrossRefGoogle Scholar
  11. Ogura K, Goshima Y. The autophagy-related kinase UNC-51 and its binding partner UNC-14 regulate the subcellular localization of the Netrin receptor UNC-5 in Caenorhabditis elegans. Development. 2006;133:3441–50.PubMedCrossRefGoogle Scholar
  12. Pankiv S, Alemu EA, Brech A, Bruun JA, Lamark T, Overvatn A, Bjørkøy G, Johansen T. FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end-directed vesicle transport. J Cell Biol. 2010;188:253–69.PubMedPubMedCentralCrossRefGoogle Scholar
  13. Tabata K, Matsunaga K, Sakane A, Sasaki T, Noda T, Yoshimori T. Rubicon and PLEKHM1 negatively regulate the endocytic/autophagic pathway via a novel Rab7-binding domain. Mol Biol Cell. 2010;21:4162–72.PubMedPubMedCentralCrossRefGoogle Scholar
  14. Terawaki S, Camosseto V, Prete F, Wenger T, Papadopoulos A, Rondeau C, Combes A, Rodriguez Rodrigues C, Vu Manh TP, Fallet M, English L, Santamaria R, Soares AR, Weil T, Hammad H, Desjardins M, Gorvel JP, Santos MA, Gatti E, Pierre P. RUN and FYVE domain-containing protein 4 enhances autophagy and lysosome tethering in response to Interleukin-4. J Cell Biol. 2015;210:1133–52.PubMedPubMedCentralCrossRefGoogle Scholar
  15. Terawaki S, Camosseto V, Pierre P, Gatti E. RUFY4: immunity piggybacking on autophagy? Autophagy. 2016;12:598–600.PubMedPubMedCentralCrossRefGoogle Scholar
  16. Wang G, Zhang Q, Song Y, Wang X, Guo Q, Zhang J, Li J, Han Y, Miao Z, Li F. PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion. Cell Death Dis. 2015;6:e1682.PubMedPubMedCentralCrossRefGoogle Scholar
  17. Wei Z, Sun M, Liu X, Zhang J, Jin Y. Rufy3, a protein specifically expressed in neurons, interacts with actin-bundling protein Fascin to control the growth of axons. J Neurochem. 2014;130:678–92.PubMedCrossRefGoogle Scholar
  18. Yamamoto H, Koga H, Katoh Y, Takahashi S, Nakayama K, Shin HW. Functional cross-talk between Rab14 and Rab4 through a dual effector, RUFY1/Rabip4. Mol Biol Cell. 2010;21:2746–55.PubMedPubMedCentralCrossRefGoogle Scholar
  19. Yang J, Kim O, Wu J, Qiu Y. Interaction between tyrosine kinase Etk and a RUN domain- and FYVE domain-containing protein RUFY1. A possible role of ETK in regulation of vesicle trafficking. J Biol Chem. 2002;277:30219–26.PubMedCrossRefGoogle Scholar
  20. Yoshida H, Okumura N, Kitagishi Y, Shirafuji N, Matsuda S. Rab5(Q79L) interacts with the carboxyl terminus of RUFY3. Int J Biol Sci. 2010;6:187–9.PubMedPubMedCentralCrossRefGoogle Scholar
  21. Yoshida H, Kitagishi Y, Okumura N, Murakami M, Nishimura Y, Matsuda S. How do you RUN on? FEBS Lett. 2011;585:1707–10.PubMedCrossRefGoogle Scholar
  22. Zheng Z, Liebers M, Zhelyazkova B, Cao Y, Panditi D, Lynch KD, Chen J, Robinson HE, Shim HS, Chmielecki J, Pao W, Engelman JA, Iafrate AJ, Le LP. Anchored multiplex PCR for targeted next-generation sequencing. Nat Med. 2014;20:1479–84.PubMedCrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  • Mayuko Ichimura
    • 1
  • Atsuko Nakanishi
    • 1
  • Yasuko Kitagishi
    • 1
  • Satoru Matsuda
    • 1
  1. 1.Department of Food Science and NutritionNara Women’s UniversityNaraJapan