Terminating protein synthesis on the ribosome requires the presence of a class I polypeptide chain release factor (RF) to induce peptidyl-tRNA hydrolysis. Bacteria possess two class I RFs; RF1 which recognises UAG and UAA, and RF2 which recognises UGA and UAA. Mitochondrial RFs are related structurally and functionally to those of bacteria. Eukaryotes posses only a single class 1 factor, eRF1, which recognises all three termination codons. Similarly, in all archaeal species where the complete sequence of the genome is available, only a single class I factor, aRF1, has been identified so far. The aRF1 family is highly homologous to the eRF1 family, indicating a common origin and ancestor molecule. The bacterial and mitochondrial class I RFs show no significant sequence similarity with their eukaryotic and archaeal counterparts and are considered to form a separate family. For more information see [, , ].This entry represents the archaeal release factor aRF1, which seems to have functional resemblance to eukaryotic RF1 [].
This fibronectin type III domain is found in fungal chitin biosynthesis protein CHS5 where, together with the neighbouring BRCT domain (), it binds to the Arf1 GTPase [].
This entry represents a group of plant ADP-ribosylation factor GTPase-activating proteins (ArfGAPs), such as AGD6/7 from Arabidopsis. ArfGAPs are a family of proteins containing an ArfGAP catalytic domain that induces the hydrolysis of GTP bound to the small guanine nucleotide-binding protein Arf, a member of the Ras superfamily of GTPases. AGD7 interacts with Arf1 and stimulates Arf1 GTPase activity in a phosphatidic acid-dependent manner. It is localised to the Golgi complex and may play a critical role in protein trafficking by controlling Arf1 activity [].
Monensin-resistant homologue 2 (Mon2) is a peripheral membrane protein involved in multiple aspects of endomembrane trafficking [, ]. In Drosophila it has been described to couple endocytosis with actin remodeling []. In budding yeast, Mon2 plays a role in endocytosis and maintenance of vacuolar structure [, ]. Mon2 is distantly related to the guanine nucleotide exchange factors (GEFs) that activate Arf1 on Golgi membranes. However, Mon2 lacks the Sec7 domain that catalyses nucleotide exchange on Arf1 []. In Drosophila it has been described to couple endocytosis with actin remodeling [].
Terminating protein synthesis on the ribosome requires the presence of a class I polypeptide chain release factor (RF) to induce peptidyl-tRNA hydrolysis. Bacteria possess two class I RFs; RF1 which recognises UAG and UAA, and RF2 which recognises UGA and UAA. Mitochondrial RFs are related structurally and functionally to those of bacteria. Eukaryotes posses only a single class 1 factor, eRF1, which recognises all three termination codons. Similarly, in all archaeal species where the complete sequence of the genome is available, only a single class I factor, aRF1, has been identified so far. The aRF1 family is highly homologous to the eRF1 family, indicating a common origin and ancestor molecule. The bacterial and mitochondrial class I RFs show no significant sequence similarity with their eukaryotic and archaeal counterparts and are considered to form a separate family. For more information see [, , ].This entry represents the eRF1 and aRF1 proteins.
Proteins in this entry are archaeal homologues of the eukaryotic gene pelota (DOM34 in yeast), which functions in recognising stalled ribosomes []. In eukaryotes, eRF3 and HBS1, which are homologous to the tRNA carrier GTPase EF1-alpha, respectively bind eRF1 and Pelota. In Archaea eRF1 (aRF1) and Pelota (aPelota) homologues exist, but no orthologues of eRF3 and Hbs1 have been detected. Instead, it seems that archaeal EF1-alpha (aEF1-alpha) could serve as a carrier GTPase protein for both aRF1 and aPelota onto the ribosome [].
SMOC-2 is a ubiquitously expressed matricellular protein that enhances the response to angiogenic growth factors, mediate cell adhesion, keratinocyte migration, and metastasis [, ]. It is also associated with vitiligo and craniofacial and dental defects []. Moreover, SMOC-2 acts as an Arf1 GTPase-activating protein (GAP) that interacts with clathrin heavy chain (CHC) and clathrin assembly protein CALM and functions in the retrograde, early endosome/trans-Golgi network (TGN) pathway in a clathrin- and AP-1-dependent manner []. SMOC-2 contains a follistatin-like (FS) domain, two thyroglobulin-like (TY) domains and an extracellular calcium-binding (EC) domain with two EF-hand calcium-binding motifs. This entry represents the EC domain [].
This entry represents a HDS (homology downstream of Sec7) domain found towards the C-terminal of guanine nucleotide exchange factors involved Golgi transport, such as budding yeast protein Sec7, protein Mon2 and BIG1-like proteins [, ]. Sec7 is involved in the secretory pathway as a protein binding scaffold for the COPII-COPI protein switch for maturation of the VTC intermediate compartments for Golgi compartment biogenesis []. Sec7 has four conserved HDS1-4 domains which act to integrate the signals from several small GTPases, including Arf1 itself, to switch Sec7 from a strongly autoinhibited to a strongly auto activated form [].
The Legionella pneumophila protein RalF is a guanine nucleotide exchange factor (GEF) of ADP-ribosylation factors (Arfs), activating and recruiting host Arf1 to the Legionella-containing vacuole []. RalF contains an N-terminal Sec7 domain and a C-terminal domain known as Sec7-capping domain (SCD). The Sec7 domain of RalF has the same overall folded structure seen in eukaryotic Sec7 domains. The eukaryotic Sec7 domain is a catalytic domain found in guanine nucleotide exchange factors (GEFs) that is sufficient to activate Arf by stimulating GDP/GTP exchange []. Structural studies showed that the C-terminal domain of RalF associates with the Sec7 domain to block access to the Arf-binding site []. This entry represents the C-terminal domain of RalF [].
This entry represent the HUS regulatory domain found towards the N terminus in guanine nucleotide exchange factors involved Golgi transport, such as budding yeast protein Sec7, protein Mon2 and BIG1-like proteins [, ].Sec7 and its homologues are guanine nucleotide exchange factors (GEFs) involved in the secretory pathway []. The full-length Sec7 functions proximally in the secretory pathway as a protein binding scaffold for the coat protein complexes COPII-COPI []. The COPII-COPI-protein switch is necessary for maturation of the vesicular-tubular cluster, VTC, intermediate compartments for Golgi compartment biogenesis. This N-terminal domain however does not appear to be binding either of the COP or the ARF [].Mon2 is distantly related to the Arf1 guanine nucleotide exchange factors (GEFs), such as Sec7. However, it lacks the Sec7 domain that catalyses nucleotide exchange on Arf1. Instead, Mon2 acts as a scaffold to recruit the Golgi-localised pool of Dop1 [].
ASAP1 is an Arf GTPase activating protein (GAP) with activity towards Arf1 and Arf5 but not Arf6 However, it has been shown to bind GTP-Arf6 stably without GAP activity []. It has been implicated in cell growth, migration, and survival, as well as in tumor invasion and malignancy. It binds paxillin and cortactin, two components of invadopodia which are essential for tumor invasiveness. It also binds focal adhesion kinase (FAK) and the SH2/SH3 adaptor CrkL [, ]. ASAP1 contains an N-terminal BAR domain, followed by a Pleckstrin homology (PH) domain, an Arf GAP domain, ankyrin (ANK) repeats, and a C-terminal SH3 domain [].This entry represents the BAR domain of ASAP1. BAR domains form dimers that bind to membranes, induce membrane bending and curvature, and may also be involved in protein-protein interactions. The BAR domain of ASAP1 mediates membrane bending, is essential for function, and autoinhibits GAP activity by interacting with the PH and/or Arf GAP domains [].
Arf GTPases are involved in the formation of coated carrier vesicles by recruiting coat proteins. This entry includes Arf1, Arf2, Arf3, Arf4, Arf5, and related proteins. Each contains an N-terminal myristoylated amphipathic helix that is folded into the protein in the GDP-bound state. GDP/GTP exchange exposes the helix, which anchors to the membrane. Following GTP hydrolysis, the helix dissociates from the membrane and folds back into the protein. A general feature of Arf1-5 signaling may be the cooperation of two Arfs at the same site. Arfs1-5 are generally considered to be interchangeable in function and location, but some specific functions have been assigned []. Arf1 localizes to the early/cis-Golgi, where it is activated by GBF1 and recruits the coat protein COPI. It also localizes to the trans-Golgi network (TGN), where it is activated by BIG1/BIG2 and recruits the AP1, AP3, AP4, and GGA proteins []. Humans, but not rodents and other lower eukaryotes, lack Arf2. Human Arf3 shares 96% sequence identity with Arf1 and is believed to generally function interchangeably with Arf1. Human Arf4 in the activated (GTP-bound) state has been shown to interact with the cytoplasmic domain of epidermal growth factor receptor (EGFR) and mediate the EGF-dependent activation of phospholipase D2 (PLD2), leading to activation of the activator protein 1 (AP-1) transcription factor []. Arf4 has also been shown to recognise the C-terminal sorting signal of rhodopsin and regulate its incorporation into specialised post-Golgi rhodopsin transport carriers (RTCs) []. There is some evidence that Arf5 functions at the early-Golgi and the trans-Golgi to affect Golgi-associated alpha-adaptin homology Arf-binding proteins (GGAs) [].
This domain is found at the N terminus of fungal chitin biosynthesis protein Chs5 []. It functions as a dimerisation domain [].Chs5/6 is a multi-protein complex conserved in fungi that interacts with chitin synthase III (Chs3p) and is involved in its transport to the cell surface from the trans-Golgi network, functioning as an exomer cargo adapter. Chs5p appears to form a complex with Chs6p and its paralogues Bch1p, Bud7p, and Bch2p. In this complex, Chs5p may act as a central scaffold []. The N-terminal domain characterized by this model forms a homodimer and has been shown to interact with Chs6p and Bch1p. It may function as a flexible hinge domain that allows the exomer to interact with both proteins and the Golgi membrane as the latter undergoes changes in curvature during the formation of transport vesicles []. The dimerization domain sits N-terminally to a conserved FBE (FN3-BRCT) unit, which binds Arf1 an is involved in the recruitment of the exomer to the membrane [].
Small GTPases form an independent superfamily within the larger class of regulatory GTP hydrolases. This superfamily contains proteins that control a vast number of important processes and possess a common, structurally preserved GTP-binding domain [, ]. Sequence comparisons of small G proteins from various species have revealed that they are conserved in primary structures at the level of 30-55% similarity [].Crystallographic analysis of various small G proteins revealed the presence of a 20kDa catalytic domain that is unique for the whole superfamily [, ]. The domain is built of five alpha helices (A1-A5), six β-strands (B1-B6) and five polypeptide loops (G1-G5). A structural comparison of the GTP- and GDP-bound form, allows one to distinguish two functional loop regions: switch I and switch II that surround the gamma-phosphate group of the nucleotide. The G1 loop (also called the P-loop) that connects the B1 strand and the A1 helix is responsible for the binding of the phosphate groups. The G3 loop provides residues for Mg2 and phosphate binding and is located at the N terminus of the A2 helix. The G1 and G3 loops are sequentially similar to Walker A and Walker B boxes that are found in other nucleotide binding motifs. The G2 loop connects the A1 helix and the B2 strand and contains a conserved Thr residue responsible for Mg2 binding. The guanine base is recognised by the G4 and G5 loops. The consensus sequence NKXD of the G4 loop contains Lys and Asp residues directly interacting with the nucleotide. Part of the G5 loop located between B6 and A5 acts as a recognition site for the guanine base [].The small GTPase superfamily can be divided into at least 8 different families, including:Arf small GTPases. GTP-binding proteins involved in protein trafficking by modulating vesicle budding and uncoating within the Golgi apparatus.Ran small GTPases. GTP-binding proteins involved in nucleocytoplasmic transport. Required for the import of proteins into the nucleus and also for RNA export.Rab small GTPases. GTP-binding proteins involved in vesicular traffic.Rho small GTPases. GTP-binding proteins that control cytoskeleton reorganisation.Ras small GTPases. GTP-binding proteins involved in signalling pathways.Sar1 small GTPases. Small GTPase component of the coat protein complex II (COPII) which promotes the formation of transport vesicles from the endoplasmic reticulum (ER).Mitochondrial Rho (Miro). Small GTPase domain found in mitochondrial proteins involved in mitochondrial trafficking.Roc small GTPases domain. Small GTPase domain always found associated with the COR domain.This entry represents a branch of the small GTPase superfamily that includes the ADP ribosylation factor Arf, Arl (Arf-like), Arp(Arf-related proteins) and the remotely related Sar (Secretion-associated and Ras-related) proteins. Arf proteins are major regulators of vesicle biogenesis in intracellular traffic []. They cycle between inactive GDP-bound and active GTP-bound forms that bind selectively to effectors. The classical structural GDP/GTP switch is characterised by conformational changes at the so-called switch 1 and switch 2 regions, which bind tightly to the gamma-phosphate of GTP but poorly or not at all to the GDP nucleotide. Structural studies of Arf1 and Arf6 have revealed that although these proteins feature the switch 1 and 2 conformational changes, they depart from other small GTP-binding proteins in that they use an additional, unique switch to propagate structural information from one side of the protein to the other. The GDP/GTP structural cycles of human Arf1 and Arf6 feature a unique conformational change that affects the beta2-beta3 strands connecting switch 1 and switch 2 (interswitch) and also the amphipathic helical N terminus. In GDP-boundArf1 and Arf6, the interswitch is retracted and forms a pocket to which the N-terminal helix binds, the latter serving as a molecular hasp to maintain the inactive conformation. In the GTP-bound form of these proteins, the interswitch undergoes a two-residue register shift that pulls switch 1 and switch 2 up, restoring an active conformation that can bind GTP. In this conformation, the interswitch projects out of the protein and extrudes the N-terminal hasp by occluding its binding pocket.
Small GTPases form an independent superfamily within the larger class of regulatory GTP hydrolases. This superfamily contains proteins that control a vast number of important processes and possess a common, structurally preserved GTP-binding domain [, ]. Sequence comparisons of small G proteins from various species have revealed that they are conserved in primary structures at the level of 30-55% similarity [].Crystallographic analysis of various small G proteins revealed the presence of a 20kDa catalytic domain that is unique for the whole superfamily [, ]. The domain is built of five alpha helices (A1-A5), six β-strands (B1-B6) and five polypeptide loops (G1-G5). A structural comparison of the GTP- and GDP-bound form, allows one to distinguish two functional loop regions: switch I and switch II that surround the gamma-phosphate group of the nucleotide. The G1 loop (also called the P-loop) that connects the B1 strand and the A1 helix is responsible for the binding of the phosphate groups. The G3 loop provides residues for Mg2 and phosphate binding and is located at the N terminus of the A2 helix. The G1 and G3 loops are sequentially similar to Walker A and Walker B boxes that are found in other nucleotide binding motifs. The G2 loop connects the A1 helix and the B2 strand and contains a conserved Thr residue responsible for Mg2 binding. The guanine base is recognised by the G4 and G5 loops. The consensus sequence NKXD of the G4 loop contains Lys and Asp residues directly interacting with the nucleotide. Part of the G5 loop located between B6 and A5 acts as a recognition site for the guanine base [].The small GTPase superfamily can be divided into at least 8 different families, including:Arf small GTPases. GTP-binding proteins involved in protein trafficking by modulating vesicle budding and uncoating within the Golgi apparatus.Ran small GTPases. GTP-binding proteins involved in nucleocytoplasmic transport. Required for the import of proteins into the nucleus and also for RNA export.Rab small GTPases. GTP-binding proteins involved in vesicular traffic.Rho small GTPases. GTP-binding proteins that control cytoskeleton reorganisation.Ras small GTPases. GTP-binding proteins involved in signalling pathways.Sar1 small GTPases. Small GTPase component of the coat protein complex II (COPII) which promotes the formation of transport vesicles from the endoplasmic reticulum (ER).Mitochondrial Rho (Miro). Small GTPase domain found in mitochondrial proteins involved in mitochondrial trafficking.Roc small GTPases domain. Small GTPase domain always found associated with the COR domain.This entry represents a branch of the small GTPase superfamily that includes the ADP ribosylation factor Arf, Arl (Arf-like), and Arp(Arf-related proteins). Arf proteins are major regulators of vesicle biogenesis in intracellular traffic []. They cycle between inactive GDP-bound and active GTP-bound forms that bind selectively to effectors. The classical structural GDP/GTP switch is characterised by conformational changes at the so-called switch 1 and switch 2 regions, which bind tightly to the gamma-phosphate of GTP but poorly or not at all to the GDP nucleotide. Structural studies of Arf1 and Arf6 have revealed that although these proteins feature the switch 1 and 2 conformational changes, they depart from other small GTP-binding proteins in that they use an additional, unique switch to propagate structural information from one side of the protein to the other. The GDP/GTP structural cycles of human Arf1 and Arf6 feature a unique conformational change that affects the beta2-beta3 strands connecting switch 1 and switch 2 (interswitch) and also the amphipathic helical N terminus. In GDP-boundArf1 and Arf6, the interswitch is retracted and forms a pocket to which the N-terminal helix binds, the latter serving as a molecular hasp to maintain the inactive conformation. In the GTP-bound form of these proteins, the interswitch undergoes a two-residue register shift that pulls switch 1 and switch 2 up, restoring an active conformation that can bind GTP. In this conformation, the interswitch projects out of the protein and extrudes the N-terminal hasp by occluding its binding pocket.
