Sly1 protein bound to Golgi syntaxin Sed5p allows assembly and contributes to specificity of SNARE fusion complexes

Fusion of transport vesicles with their target organelles involves specific membrane proteins, SNAREs, which form tight complexes bridging the membranes to be fused. Evidence from yeast and mammals indicates that Sec1 family proteins act as regulators of membrane fusion by binding to the target membrane SNAREs. In experiments with purified proteins, we now made the observation that the ER to Golgi core SNARE fusion complex could be assembled on syntaxin Sed5p tightly bound to the Sec1-related Sly1p. Sly1p also bound to preassembled SNARE complexes in vitro and was found to be part of a vesicular/target membrane SNARE complex immunoprecipitated from yeast cell lysates. This is in marked contrast to the exocytic SNARE assembly in neuronal cells where high affinity binding of N-Sec1/Munc-18 to syntaxin 1A precluded core SNARE fusion complex formation. We also found that the kinetics of SNARE complex formation in vitro with either Sly1p-bound or free Sed5p was not significantly different. Importantly, several presumably nonphysiological SNARE complexes easily generated with Sed5p did not form when the syntaxin was first bound to Sly1p. This indicates for the first time that a Sec1 family member contributes to the specificity of SNARE complex assembly.     

Multiple SNARE interactions of an SM protein: Sed5p/Sly1p binding is dispensable for transport

Sec1/Munc18 (SM) proteins are central to intracellular transport and neurotransmitter release but their exact role is still elusive. Several SM proteins, like the neuronal N-Sec1 and the yeast Sly1 protein, bind their cognate t-SNAREs with high affinity. This has been thought to be critical for their function. Here, we show that various mutant forms of Sly1p and the Golgi-localized syntaxin Sed5p, which abolish their high-affinity interaction, are fully functional in vivo, indicating that the tight interaction of the two molecules per se is not relevant for proper function. Mutant Sly1p unable to bind Sed5p is excluded from core SNARE complexes, also demonstrating that Sly1p function is not directly coupled to assembled SNARE complexes thought to execute membrane fusion. We also find that wild-type Sly1p and mutant Sly1p unable to bind Sed5p directly interact with selected ER-to-Golgi and intra-Golgi nonsyntaxin SNAREs. The newly identified, direct interactions of the SM protein with nonsytaxin SNAREs might provide a molecular mechanism by which SNAREs can be activated to engage in pairing and assemble into fusogenic SNARE complexes.     

Structural basis for ARF1-mediated recruitment of ARHGAP21 to Golgi membranes

ARHGAP21 is a Rho family GTPase-activating protein (RhoGAP) that controls the Arp2/3 complex and F-actin dynamics at the Golgi complex by regulating the activity of the small GTPase Cdc42. ARHGAP21 is recruited to the Golgi by binding to another small GTPase, ARF1. Here, we present the crystal structure of the activated GTP-bound form of ARF1 in a complex with the Arf-binding domain (ArfBD) of ARHGAP21 at 2.1 A resolution. We show that ArfBD comprises a PH domain adjoining a C-terminal alpha helix, and that ARF1 interacts with both of these structural motifs through its switch regions and triggers structural rearrangement of the PH domain. We used site-directed mutagenesis to confirm that both the PH domain and the helical motif are essential for the binding of ArfBD to ARF1 and for its recruitment to the Golgi. Our data demonstrate that two well-known small GTPase-binding motifs, the PH domain and the alpha helical motif, can combine to create a novel mode of binding to Arfs.     

Multiple features within the syntaxin Sed5p mediate its Golgi localization

Protein retention and the transport of proteins and lipids into and out of the Golgi is intimately linked to the biogenesis and homeostasis of this sorting hub of eukaryotic cells. Of particular importance are membrane proteins that mediate membrane fusion events with and within the Golgi—the Soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs). In the Golgi of budding yeast cells, the syntaxin SNARE Sed5p oversees membrane fusion events. Determining how Sed5p is localized to and trafficked within the Golgi is critical to informing our understanding of the mechanism(s) of biogenesis and homeostasis of this organelle. Here we establish that the steady‐state localization of Sed5p to the Golgi appears to be primarily conformation‐based relying on intra‐molecular associations between the Habc domain and SNARE‐motif while its tribasic COPI‐coatomer binding motif plays a role in intra‐Golgi retention.     

Structural basis of wedging the Golgi membrane by FAPP pleckstrin homology domains

The mechanisms underlying Golgi targeting and vesiculation are unknown, although the responsible phosphatidylinositol 4‐phosphate (PtdIns(4)P) ligand and four‐phosphate‐adaptor protein (FAPP) modules have been defined. The micelle‐bound structure of the FAPP1 pleckstrin homology domain reveals how its prominent wedge independently tubulates Golgi membranes by leaflet penetration. Mutations compromising the exposed hydrophobicity of full‐length FAPP2 abolish lipid monolayer binding and compression. The trafficking process begins with an electrostatic approach, phosphoinositide sampling and perpendicular penetration. Extensive protein contacts with PtdIns(4)P and neighbouring phospholipids reshape the bilayer and initiate tubulation through a conserved wedge with features shared by diverse protein modules.     

Control of Golgi morphology and function by Sed5 t-SNARE phosphorylation

Previously, we demonstrated that the phosphorylation of t-SNAREs by protein kinase A (PKA) affects their ability to participate in SNARE complexes and to confer endocytosis and exocytosis in yeast. Here, we show that the presumed phosphorylation of a conserved membrane-proximal PKA consensus site (serine-317) in the Sed5 t-SNARE regulates endoplasmic reticulum (ER)-Golgi transport, as well as Golgi morphology. Sed5 is a phosphoprotein, and both alanine and aspartate substitutions in serine-317 directly affect intracellular protein trafficking. The aspartate substitution results in elaboration of the ER, defects in Golgi-ER retrograde transport, an accumulation of small transport vesicles, and the inhibition of growth of most cell types. In contrast, the alanine substitution has no deleterious effects upon transport and growth, but results in ordering of the Golgi into a structure reminiscent of mammalian apparatus. This structure seems to require the recycling of Sed5, because it was found not to occur in sec21-2 cells that are defective in retrograde transport. Thus, a cycle of Sed5 phosphorylation and dephosphorylation is required for normal t-SNARE function and may choreograph Golgi ordering and dispersal.     

Structural basis for recruitment of human flap endonuclease 1 to PCNA

Flap endonuclease-1 (FEN1) is a key enzyme for maintaining genomic stability and replication. Proliferating cell nuclear antigen (PCNA) binds FEN1 and stimulates its endonuclease activity. The structural basis of the FEN1-PCNA interaction was revealed by the crystal structure of the complex between human FEN1 and PCNA. The main interface involves the C-terminal tail of FEN1, which forms two beta-strands connected by a short helix, the betaA-alphaA-betaB motif, participating in beta-beta and hydrophobic interactions with PCNA. These interactions are similar to those previously observed for the p21CIP1/WAF1 peptide. However, this structure involving the full-length enzyme has revealed additional interfaces that are involved in the core domain. The interactions at the interfaces maintain the enzyme in an inactive 'locked-down' orientation and might be utilized in rapid DNA-tracking by preserving the central hole of PCNA for sliding along the DNA. A hinge region present between the core domain and the C-terminal tail of FEN1 would play a role in switching the FEN1 orientation from an inactive to an active orientation.     

Structural basis for recruitment of BRCA2 by PALB2

The breast cancer 2, early onset protein (BRCA2) is central to the repair of DNA damage by homologous recombination. BRCA2 recruits the recombinase RAD51 to sites of damage, regulates its assembly into nucleoprotein filaments and thereby promotes homologous recombination. Localization of BRCA2 to nuclear foci requires its association with the partner and localizer of BRCA2 (PALB2), mutations in which are associated with cancer predisposition, as well as subtype N of Fanconi anaemia. We have determined the structure of the PALB2 carboxy‐terminal β‐propeller domain in complex with a BRCA2 peptide. The structure shows the molecular determinants of this important protein–protein interaction and explains the effects of both cancer‐associated truncating mutants in PALB2 and missense mutations in the amino‐terminal region of BRCA2.     

Structural basis for recruitment of GRIP domain golgin-245 by small GTPase Arl1

Recruitment of the GRIP domain golgins to the trans-Golgi network is mediated by Arl1, a member of the ARF/Arl small GTPase family, through interaction between their GRIP domains and Arl1-GTP. The crystal structure of Arl1-GTP in complex with the GRIP domain of golgin-245 shows that Arl1-GTP interacts with the GRIP domain predominantly in a hydrophobic manner, with the switch II region conferring the main recognition surface. The involvement of the switch and interswitch regions in the interaction between Arl1-GTP and GRIP accounts for the specificity of GRIP domain for Arl1-GTP. Mutations that abolished the Arl1-mediated Golgi localization of GRIP domain golgins have been mapped on the interface between Arl1-GTP and GRIP. Notably, the GRIP domain forms a homodimer in which each subunit interacts separately with one Arl1-GTP. Mutations disrupting the GRIP domain dimerization also abrogated its Golgi targeting, suggesting that the dimeric form of GRIP domain is a functional unit.     

Structural basis for recruitment of the CHK1 DNA damage kinase by the CLASPIN scaffold protein

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Structural basis for the accessory protein recruitment by the gamma-adaptin ear domain

The adaptor proteins AP-1 and GGA regulate membrane traffic between the trans-Golgi network (TGN) and endosomes/lysosomes through ARF-regulated membrane association, recognition of sorting signals, and recruitment of clathrin and accessory proteins. The gamma 1-adaptin subunits of AP-1 and GGA possess homologous ear domains involved in the recruitment of accessory proteins, gamma-synergin and Rabaptin-5. The crystal structure of the human gamma 1-adaptin ear domain consists solely of an immunoglobulin-like fold, unlike the alpha-adaptin ear domain. Structure-based mutational analyses reveal a binding site for the accessory proteins that is composed of conserved basic residues, indicating that the recruitment mechanism in gamma 1-adaptin and GGA is distinct from that in alpha-adaptin.     

Structural basis for the recruitment of ERCC1-XPF to nucleotide excision repair complexes by XPA

The nucleotide excision repair (NER) pathway corrects DNA damage caused by sunlight, environmental mutagens and certain antitumor agents. This multistep DNA repair reaction operates by the sequential assembly of protein factors at sites of DNA damage. The efficient recognition of DNA damage and its repair are orchestrated by specific protein-protein and protein-DNA interactions within NER complexes. We have investigated an essential protein-protein interaction of the NER pathway, the binding of the XPA protein to the ERCC1 subunit of the repair endonuclease ERCC1-XPF. The structure of ERCC1 in complex with an XPA peptide shows that only a small region of XPA interacts with ERCC1 to form a stable complex exhibiting submicromolar binding affinity. However, this XPA peptide is a potent inhibitor of NER activity in a cell-free assay, blocking the excision of a cisplatin adduct from DNA. The structure of the peptide inhibitor bound to its target site reveals a binding interface that is amenable to the development of small molecule peptidomimetics that could be used to modulate NER repair activities in vivo.     

Structural basis for recruitment of RILP by small GTPase Rab7

Rab7 regulates vesicle traffic from early to late endosomes, and from late endosomes to lysosomes. The crystal structure of Rab7-GTP in complex with the Rab7 binding domain of RILP reveals that Rab7 interacts with RILP specifically via two distinct areas, with the first one involving the switch and interswitch regions and the second one consisting of RabSF1 and RabSF4. Disruption of these interactions by mutations abrogates late endosomal/lysosomal targeting of Rab7 and RILP. The Rab7 binding domain of RILP forms a coiled-coil homodimer with two symmetric surfaces to interact with two separate Rab7-GTP molecules, forming a dyad configuration of Rab7-RILP(2)-Rab7. Mutations that disrupt RILP dimerization also abolish its interactions with Rab7-GTP and late endosomal/lysosomal targeting, suggesting that the dimeric form of RILP is a functional unit. Structural comparison suggests that the combined use of RabSF1 and RabSF4 with the switch regions may be a general mode of action for most Rab proteins in regulating membrane trafficking.     

Structural basis for Arl1-dependent targeting of homodimeric GRIP domains to the Golgi apparatus

Golgins are large coiled-coil proteins that play a role in Golgi structure and vesicle traffic. The Arf-like GTPase Arl1 regulates the translocation of GRIP domain-containing golgins to Golgi membranes. We report here the 1.7 A resolution structure of human Arl1-GTP in a complex with the GRIP domain of golgin-245. The structure reveals that the GRIP domain consists of an S-shaped arrangement of three helices. The domain forms a homodimer that binds two Arl1-GTPs using two helices from each monomer. The structure is consistent with golgin-245 forming parallel coiled-coils and suggests how Arl1-GTP/GRIP complexes interact with Golgi membranes via the N termini of Arl1-GTP and the C-terminal tails of the GRIP domains. In cells, bivalent association with Arl1-GTP would increase residence time of the golgins on Golgi membranes. Despite no conservation of sequence, topology, or even helical direction, several other effectors form similar interactions with small GTPases via a pair of alpha helices, suggesting a common structural basis for effector recognition.     

Structural basis for endosomal recruitment of ESCRT-I by ESCRT-0 in yeast

The ESCRT‐0 and ESCRT‐I complexes coordinate the clustering of ubiquitinated cargo with intralumenal budding of the endosomal membrane, two essential steps in vacuolar/lysosomal protein sorting from yeast to humans. The 1.85‐Å crystal structure of interacting regions of the yeast ESCRT‐0 and ESCRT‐I complexes reveals that PSDP motifs of the Vps27 ESCRT‐0 subunit bind to a novel electropositive N‐terminal site on the UEV domain of the ESCRT‐I subunit Vps23 centred on Trp16. This novel site is completely different from the C‐terminal part of the human UEV domain that binds to P(S/T)AP motifs of human ESCRT‐0 and HIV‐1 Gag. Disruption of the novel PSDP‐binding site eliminates the interaction in vitro and blocks enrichment of Vps23 in endosome‐related class E compartments in yeast cells. However, this site is non‐essential for sorting of the ESCRT cargo Cps1. Taken together, these results show how a conserved motif/domain pair can evolve to use strikingly different binding modes in different organisms.     

Proteins in the early golgi compartment of Saccharomyces cerevisiae immunoisolated by Sed5p

The yeast tSNARE Sed5p is considered to mainly reside in the early Golgi compartment at the steady state of its intracellular cycling. To better understand this compartment, we immunoisolated a membrane subfraction having Sed5p on the surface (the Sed5 vesicles). Immunoblot studies showed that considerable portions (20-30%) of the Golgi mannosyltransferases (Mnt1p, Van1p, and Mnn9p) were simultaneously recovered while the late Golgi (Kex2p) or endoplasmic reticulum (Sec71p) proteins were almost excluded. The N-terminal sequences of the polypeptides detectable by Coomassie blue staining indicated that the prominent components of the Sed5 vesicles include Anp1p, Emp24p, Erv25p, Erp1p, Ypt52p, and a putative membrane protein of unknown function (Yml067c).     

Structural basis for ESCRT-III CHMP3 recruitment of AMSH

Endosomal sorting complexes required for transport (ESCRT) recognize ubiquitinated cargo and catalyze diverse budding processes including multivesicular body biogenesis, enveloped virus egress, and cytokinesis. We present the crystal structure of an N-terminal fragment of the deubiquitinating enzyme AMSH (AMSHΔC) in complex with the C-terminal region of ESCRT-III CHMP3 (CHMP3ΔN). AMSHΔC folds into an elongated 90?? long helical assembly that includes an unusual MIT domain. CHMP3ΔN is unstructured in solution and helical in complex with AMSHΔC, revealing a novel MIT domain interacting motif (MIM) that does not overlap with the CHMP1-AMSH binding site. ITC and SPR measurements demonstrate an unusual high-affinity MIM-MIT interaction. Structural analysis suggests a regulatory role for the N-terminal helical segment of AMSHΔC and its destabilization leads to a loss of function during HIV-1 budding. Our results indicate a tight coupling of ESCRT-III CHMP3 and AMSH functions and provide insight into the regulation of ESCRT-III.