The Dsl1 tethering complex actively participates in soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) complex assembly at the endoplasmic reticulum in Saccharomyces cerevisiae

Intracellular transport is largely dependent on vesicles that bud off from one compartment and fuse with the target compartment. The first contact of an incoming vesicle with the target membrane is mediated by tethering factors. The tethering factor responsible for recruiting Golgi-derived vesicles to the ER is the Dsl1 tethering complex, which is comprised of the essential proteins Dsl1p, Dsl3p, and Tip20p. We investigated the role of the Tip20p subunit at the ER by analyzing two mutants, tip20-5 and tip20-8. Both mutants contained multiple mutations that were scattered throughout the TIP20 sequence. Individual mutations could not reproduce the temperature-sensitive phenotype of tip20-5 and tip20-8, indicating that the overall structure of Tip20p might be altered in the mutants. Using molecular dynamics simulations comparing Tip20p and Tip20-8p revealed that some regions, particularly the N-terminal domain and parts of the stalk region, were more flexible in the mutant protein, consistent with its increased susceptibility to proteolysis. Both Tip20-5p and Tip20-8p mutants prevented proper ER trans-SNARE complex assembly in vitro. Moreover, Tip20p mutant proteins disturbed the interaction between Dsl1p and the coatomer coat complex, indicating that the Dsl1p-coatomer interaction could be stabilized or regulated by Tip20p. We provide evidence for a direct role of the Dsl1 complex, in particular Tip20p, in the formation and stabilization of ER SNARE complexes.     

Dsl1p, Tip20p, and the novel Dsl3(Sec39) protein are required for the stability of the Q/t-SNARE complex at the endoplasmic reticulum in yeast

The "Dsl1p complex" in Saccharomyces cerevisiae, consisting of Dsl1p and Tip20p, is involved in Golgi-ER retrograde transport and it is functionally conserved from yeast to mammalian cells. To further characterize this complex, we analyzed the function of Dsl3p, a protein that interacts with Dsl1p in yeast two hybrids screens. DSL3, recently identified in a genome wide analysis of essential genes as SEC39, encodes a cytosolic protein of 82 kDa that is peripherally associated with membranes derived from the ER. There is strong genetic interaction between DSL3 and other factors required for Golgi-ER retrograde transport. Size exclusion chromatography and affinity purification approaches confirmed that Dsl3p is associated with subunits of the "Dsl1p complex." The complex also includes the Q/t-SNARE proteins, Use1p, Sec20p, and Ufe1p, integral membrane proteins that constitute the trimeric acceptor for R/v-SNAREs on Golgi-derived vesicles at the ER. Using mutants, we performed a detailed analysis of interactions between subunits of the Dsl1p complex and the ER-localized SNARE proteins. This analysis showed that both Dsl1p and Dsl3p are required for the stable interaction of the SNARE Use1p with a central subcomplex consisting of Tip20p and the SNARE proteins Ufe1p and Sec20p.     

Golgi-to-Endoplasmic Reticulum (ER) Retrograde Traffic in Yeast Requires Dsl1p, a Component of the ER Target Site that Interacts with a COPI Coat Subunit

DSL1 was identified through its genetic interaction with SLY1, which encodes a t-SNARE-interacting protein that functions in endoplasmic reticulum (ER)-to-Golgi traffic. Conditional dsl1 mutants exhibit a block in ER-to-Golgi traffic at the restrictive temperature. Here, we show that dsl1 mutants are defective for retrograde Golgi-to-ER traffic, even under conditions where no anterograde transport block is evident. These results suggest that the primary function of Dsl1p may be in retrograde traffic, and that retrograde defects can lead to secondary defects in anterograde traffic. Dsl1p is an ER-localized peripheral membrane protein that can be extracted from the membrane in a multiprotein complex. Immunoisolation of the complex yielded Dsl1p and proteins of approximately 80 and approximately 55 kDa. The approximately 80-kDa protein has been identified as Tip20p, a protein that others have shown to exist in a tight complex with Sec20p, which is approximately 50 kDa. Both Sec20p and Tip20p function in retrograde Golgi-to-ER traffic, are ER-localized, and bind to the ER t-SNARE Ufe1p. These findings suggest that an ER-localized complex of Dsl1p, Sec20p, and Tip20p functions in retrograde traffic, perhaps upstream of a Sly1p/Ufe1p complex. Last, we show that Dsl1p interacts with the delta-subunit of the retrograde COPI coat, Ret2p, and discuss possible roles for this interaction.     

The Dsl1 protein tethering complex is a resident endoplasmic reticulum complex, which interacts with five soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptors (SNAREs): implications for fusion and fusion regulation

Retrograde vesicular transport from the Golgi to the ER requires the Dsl1 tethering complex, which consists of the three subunits Dsl1, Dsl3, and Tip20. It forms a stable complex with the SNAREs Ufe1, Use1, and Sec20 to mediate fusion of COPI vesicles with the endoplasmic reticulum. Here, we analyze molecular interactions between five SNAREs of the ER (Ufe1, Use1, Sec20, Sec22, and Ykt6) and the Dsl1 complex in vitro and in vivo. Of the two R-SNAREs, Sec22 is preferred over Ykt6 in the Dsl-SNARE complex. The NSF homolog Sec18 can displace Ykt6 but not Sec22, suggesting a regulatory function for Ykt6. In addition, our data also reveal that subunits of the Dsl1 complex (Dsl1, Dsl3, and Tip20), as well as the SNAREs Ufe1 and Sec20, are ER-resident proteins that do not seem to move into COPII vesicles. Our data support a model, in which a tethering complex is stabilized at the organelle membrane by binding to SNAREs, recognizes the incoming vesicle via its coat and then promotes its SNARE-mediated fusion.     

The TIP1 gene of Saccharomyces cerevisiae encodes an 80 kDa cytoplasmic protein that interacts with the cytoplasmic domain of Sec20p.

The SEC20 gene of Saccharomyces cerevisiae encodes a 50 kDa type II integral membrane glycoprotein that is required for endoplasmic reticulum (ER) to Golgi transport. Here, we have used a genetic screen, based on the lethal effect of overexpressing the cytoplasmic domain of Sec20p, to identify a novel cytosolic factor that interacts with SEC20. This factor is an 80 kDa cytoplasmic protein encoded by the TIP1 (SEC twenty interacting protein) gene. Coimmunoprecipitation and immunofluorescence using Tip1p and Sec20p or its cytoplasmic domain showed that the two proteins physically interact to form a stable complex. Like SEC20, TIP1 is required for ER to Golgi transport and depletion of Tip1p results in accumulation of an extensive network of ER plus small transport vesicles. We therefore propose that Sec20p and Tip1p act together as a functional unit in the ER to Golgi transport step.     

Dsl1p, an essential protein required for membrane traffic at the endoplasmic reticulum/Golgi interface in yeast

To identify novel factors required for ER to Golgi transport in yeast we performed a screen for genes that, when mutated, confer a dependence on a dominant mutant form of the ER to Golgi vesicle docking factor Sly1p, termed Sly1-20p. DSL1 , a novel gene isolated in the screen, encodes an essential protein with a predicted molecular mass of 88 kDa. DSL1 is required for transport between the ER and the Golgi because strains bearing mutant alleles of this gene accumulate the pre-Golgi form of transported proteins at the restrictive temperature. Two strains bearing temperature-sensitive alleles of DSL1 display distinct phenotypes as observed by electron microscopy at the restrictive temperature; although both strains accumulate ER membrane, one also accumulates vesicles. Interestingly, the inviability of strains bearing several mutant alleles of DSL1 can be suppressed by expression of either Erv14p (a protein required for the movement of a specific protein from the ER to the Golgi), Sec21p (the γ-subunit of the COPI coat protein complex coatomer), or Sly1-20p. Because the strongest suppressor is SEC21 , we proposed that Dsl1p functions primarily in retrograde Golgi to ER traffic, although it is possible that Dsl1p functions in anterograde traffic as well, perhaps at the docking stage, as suggested by the suppression by SLY1-20 .     

Novel cargo-binding site in the beta and delta subunits of coatomer

Arginine (R)-based ER localization signals are sorting motifs that confer transient ER localization to unassembled subunits of multimeric membrane proteins. The COPI vesicle coat binds R-based signals but the molecular details remain unknown. Here, we use reporter membrane proteins based on the proteolipid Pmp2 fused to GFP and allele swapping of COPI subunits to map the recognition site for R-based signals. We show that two highly conserved stretches--in the beta- and delta-COPI subunits--are required to maintain Pmp2GFP reporters exposing R-based signals in the ER. Combining a deletion of 21 residues in delta-COP together with the mutation of three residues in beta-COP gave rise to a COPI coat that had lost its ability to recognize R-based signals, whilst the recognition of C-terminal di-lysine signals remained unimpaired. A homology model of the COPI trunk domain illustrates the recognition of R-based signals by COPI.     

COP I domains required for coatomer integrity, and novel interactions with ARF and ARF-GAP

We performed a systematic mapping of interaction domains on COP I subunits to gain novel insights into the architecture of coatomer. Using the two-hybrid system, we characterize the domain structure of the alpha-, beta'-, epsilon-COP and beta-, gamma-, delta-, zeta-COP coatomer subcomplexes and identify links between them that contribute to coatomer integrity. Our results demonstrate that the domain organization of the beta-, gamma-, delta-, zeta-COP subcomplex and AP adaptor complexes is related. Through in vivo analysis of alpha-COP truncation mutants, we characterize distinct functional domains on alpha-COP. Its N-terminal WD40 domain is dispensable for yeast cell viability and overall coatomer function, but is required for KKXX-dependent trafficking. The last approximately 170 amino acids of alpha-COP are also non-essential for cell viability, but required for epsilon-COP incorporation into coatomer and maintainance of normal epsilon-COP levels. Further, we demonstrate novel direct interactions of coatomer subunits with regulatory proteins: beta'- and gamma-COP interact with the ARF-GTP-activating protein (GAP) Glo3p, but not Gcs1p, and beta- and epsilon-COP interact with ARF-GTP. Glo3p also interacts with intact coatomer in vitro.     

Multiple and stepwise interactions between coatomer and ADP-ribosylation factor-1 (Arf1)-GTP

The small GTPase ADP-ribosylation factor-1 (Arf1) plays a key role in the formation of coat protein I (COP I)-coated vesicles. Upon recruitment to the donor Golgi membrane by interaction with dimeric p24 proteins, Arf1's GDP is exchanged for GTP. Arf1-GTP then dissociates from p24, and together with other Golgi membrane proteins, it recruits coatomer, the heptameric coat protein complex of COP I vesicles, from the cytosol. In this process, Arf1 was shown to specifically interact with the coatomer beta and gamma-COP subunits through its switch I region, and with epsilon-COP. Here, we mapped the interaction of the Arf1-GTP switch I region to the trunk domains of beta and gamma-COP. Site-directed photolabeling at position 167 in the C-terminal helix of Arf1 revealed a novel interaction with coatomer via a putative longin domain of delta-COP. Thus, coatomer is linked to the Golgi through multiple interfaces with membrane-bound Arf1-GTP. These interactions are located within the core, adaptor-like domain of coatomer, indicating an organizational similarity between the COP I coat and clathrin adaptor complexes.     

Coatomer, the coat protein of COPI transport vesicles, discriminates endoplasmic reticulum residents from p24 proteins

In the formation of COPI vesicles, interactions take place between the coat protein coatomer and membrane proteins: either cargo proteins for retrieval to the endoplasmic reticulum (ER) or proteins that cycle between the ER and the Golgi. While the binding sites on coatomer for ER residents have been characterized, how cycling proteins bind to the COPI coat is still not clear. In order to understand at a molecular level the mechanism of uptake of such proteins, we have investigated the binding to coatomer of p24 proteins as examples of cycling proteins as well as that of ER-resident cargos. The p24 proteins required dimerization to interact with coatomer at two independent binding sites in gamma-COP. In contrast, ER-resident cargos bind to coatomer as monomers and to sites other than gamma-COP. The COPI coat therefore discriminates between p24 proteins and ER-resident proteins by differential binding involving distinct subunits.     

Architecture of coatomer: molecular characterization of delta-COP and protein interactions within the complex

Coatomer is a cytosolic protein complex that forms the coat of COP I- coated transport vesicles. In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, [COPs] alpha-zeta), we engaged in a program to clone and characterize the individual coatomer subunits. We have now cloned, sequenced, and overexpressed bovine alpha-COP, the 135-kD subunit of coatomer as well as delta-COP, the 57-kD subunit and have identified a yeast homolog of delta-COP by cDNA sequence comparison and by NH2-terminal peptide sequencing. delta-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2. We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP I-coated transport vesicles and in the budding regions of the Golgi membranes. A knock-out of the delta-COP gene in yeast is lethal. Immunoprecipitation, as well as analysis exploiting the two-hybrid system in a complete COP screen, showed physical interactions between alpha- and epsilon-COPs and between beta- and delta-COPs. Moreover, the two-hybrid system indicates interactions between gamma- and zeta-COPs as well as between alpha- and beta' COPs. We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex.     

Sec20p-interacting proteins (Tip20p,Ufe1p) in the retrograde secretory pathway of the fungal pathogen<Emphasis Type="Italic"> Candida albicans</Emphasis>

Sec20p is an essential Type-II membrane protein of the human fungal pathogen Candida albicans, which is thought to be involved in mediating retrograde vesicle traffic from the Golgi to the endoplasmic reticulum (ER). Using an epitope-tagged Sec20p we obtained evidence for its localization in ER membranes, which is consistent with its proposed role in an ER-tSNARE complex. Two genes encoding potential interaction partners for Sec20p, Tip20p and Ufe1p, were identified in genomic sequences of C. albicans; these show 18% and 27% identity, respectively, to homologues in Saccharomyces cerevisiae. An interaction between the cytoplasmic domain of Sec20p and Tip20p was demonstrated by two-hybrid analysis; in addition, Tip20p was found to form homodimers. Interaction between Sec20p and Tip20p in vivo was verified by co-immunoprecipation experiments. CaUFE1, which encodes a potential ER-tSNARE, was able to complement a thermosensitive ufe1 mutation in S. cerevisiae, suggesting functional conservation between the two fungal proteins. Thus, although the sequences of some components of the ER-tSNARE complex have diverged considerably during evolution, it appears that they have retained similar functions in C. albicans and S. cerevisiae.     

COPII coat subunit interactions: Sec24p and Sec23p bind to adjacent regions of Sec16p.

Formation of COPII-coated vesicles at the endoplasmic reticulum (ER) requires assembly onto the membrane of five cytosolic coat proteins, Sec23p, Sec24p, Sec13p, Sec31p, and Sar1p. A sixth vesicle coat component, Sec16p, is tightly associated with the ER membrane and has been proposed to act as a scaffold for membrane association of the soluble coat proteins. We previously showed that Sec23p binds to the C-terminal region of Sec16p. Here we use two-hybrid and coprecipitation assays to demonstrate that the essential COPII protein Sec24p binds to the central region of Sec16p. In vitro reconstitution of binding with purified recombinant proteins demonstrates that the interaction of Sec24p with the central domain of Sec16p does not depend on the presence of Sec23p. However, Sec23p facilitates binding of Sec24p to Sec16p, and the three proteins can form a ternary complex in vitro. Truncations of Sec24p demonstrate that the N-terminal and C-terminal regions of Sec24p display different binding specificities. The C terminus binds to the central domain of Sec16p, whereas the N terminus of Sec24p binds to both the central domain of Sec16p and to Sec23p. These findings define binding to Sec16p as a new function for Sec24p and support the idea that Sec16p organizes assembly of the COPII coat.     

Identification of Discrete Sites in Yip1A Necessary for Regulation of Endoplasmic Reticulum Structure

The endoplasmic reticulum (ER) of specialized cells can undergo dramatic changes in structural organization, including formation of concentric whorls. We previously reported that depletion of Yip1A, an integral membrane protein conserved between yeast and mammals, caused ER whorl formation reminiscent of that seen in specialized cells. Yip1A and its yeast homologue Yip1p cycle between the ER and early Golgi, have been implicated in a number of distinct trafficking steps, and interact with a conserved set of binding partners including Yif1p/Yif1A and the Ypt1/Ypt31 Rab GTPases. Here, we carried out a mutational analysis of Yip1A to obtain insight into how it regulates ER whorl formation. Most of the Yip1A cytoplasmic domain was dispensable, whereas the transmembrane (TM) domain, especially residues within predicted TM helices 3 and 4, were sensitive to mutagenesis. Comprehensive analysis revealed two discrete functionally required determinants. One was E95 and flanking residues L92 and L96 within the cytoplasmic domain; the other was K146 and nearby residue V152 within the TM domain. Notably, the identified determinants correspond closely to two sites previously found to be essential for yeast viability (E76 and K130 in Yip1p corresponding to E95 and K146 in Yip1A, respectively). In contrast, a third site (E89) also essential for yeast viability (E70 in Yip1p) was dispensable for regulation of whorl formation. Earlier work showed that E76 (E95) was dispensable for binding Yif1p or Ypt1p/Ypt31p, whereas E70 (E89) was required. Collectively, these findings suggest that the ability of Yip1A to bind its established binding partners may be uncoupled from its ability to control ER whorl formation. In support, Yif1A knockdown did not cause ER whorl formation. Thus Yip1A may use the sites identified herein to interact with a novel binding partner to regulate ER membrane organization.     

Characterization of Lck-binding elements in the herpesviral regulatory Tip protein

Herpesvirus saimiri encodes a tyrosine kinase interacting protein (Tip) that binds to T-cell-specific tyrosine kinase Lck via multiple sequence motifs and controls its activity. The regulation of Lck by Tip represents a key mechanism in the transformation of human T-lymphocytes during herpesviral infection. In this study, the interaction of Tip with the regulatory SH3 and SH2 domains of Lck was investigated by biophysical and computational techniques. NMR spectroscopy of isotopically labeled Tip(140-191) revealed that the interaction with the LckSH3 domain is not restricted to the classical proline-rich motif, but also involves the C-terminally adjacent residues which pack into a hydrophobic pocket on the surface of the SH3 domain, thus playing a likely role in mediating binding specificity. Fluorescence binding studies of Tip further demonstrate that Tyr127 in its phosphorylated form represents a strong ligand of the LckSH2 domain, indicating the presence of an additional Lck interaction motif. In contrast, Tyr114, known to be essential for STAT-3 binding, does not interact with the LckSH2 domain, showing that the tyrosines in Tip exhibit distinct binding specificity. The existence of numerous interaction sites between Tip and the regulatory domains of Lck implies a complex regulatory mechanism and may have evolved to allow a gradual regulation of Lck activity in different pathogenic states.     

A Highlights from MBoC Selection: ER-associated retrograde SNAREs and the Dsl1 complex mediate an alternative,Sey1p-independent homotypic ER fusion pathway

The peripheral endoplasmic reticulum (ER) network is dynamically maintained by homotypic (ER–ER) fusion. In Saccharomyces cerevisiae, the dynamin-like GTPase Sey1p can mediate ER–ER fusion, but sey1Δ cells have no growth defect and only slightly perturbed ER structure. Recent work suggested that ER-localized soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) mediate a Sey1p-independent ER–ER fusion pathway. However, an alternative explanation—that the observed phenotypes arose from perturbed vesicle trafficking—could not be ruled out. In this study, we used candidate and synthetic genetic array (SGA) approaches to more fully characterize SNARE-mediated ER–ER fusion. We found that Dsl1 complex mutations in sey1Δ cells cause strong synthetic growth and ER structure defects and delayed ER–ER fusion in vivo, additionally implicating the Dsl1 complex in SNARE-mediated ER–ER fusion. In contrast, cytosolic coat protein I (COPI) vesicle coat mutations in sey1Δ cells caused no synthetic defects, excluding perturbed retrograde trafficking as a cause for the previously observed synthetic defects. Finally, deleting the reticulons that help maintain ER architecture in cells disrupted for both ER–ER fusion pathways caused almost complete inviability. We conclude that the ER SNAREs and the Dsl1 complex directly mediate Sey1p-independent ER–ER fusion and that, in the absence of both pathways, cell viability depends upon membrane curvature–promoting reticulons.     

ER arrival sites for COPI vesicles localize to hotspots of membrane trafficking

COPI‐coated vesicles mediate retrograde membrane traffic from the cis‐Golgi to the endoplasmic reticulum (ER) in all eukaryotic cells. However, it is still unknown whether COPI vesicles fuse everywhere or at specific sites with the ER membrane. Taking advantage of the circumstance that the vesicles still carry their coat when they arrive at the ER, we have visualized active ER arrival sites (ERAS) by monitoring contact between COPI coat components and the ER‐resident Dsl tethering complex using bimolecular fluorescence complementation (BiFC). ERAS form punctate structures near Golgi compartments, clearly distinct from ER exit sites. Furthermore, ERAS are highly polarized in an actin and myosin V‐dependent manner and are localized near hotspots of plasma membrane expansion. Genetic experiments suggest that the COPI?Dsl BiFC complexes recapitulate the physiological interaction between COPI and the Dsl complex and that COPI vesicles are mistargeted in dsl1 mutants. We conclude that the Dsl complex functions in confining COPI vesicle fusion sites.     

Drosophila Sec16 mediates the biogenesis of tER sites upstream of Sar1 through an arginine-rich motif

tER sites are specialized cup-shaped ER subdomains characterized by the focused budding of COPII vesicles. Sec16 has been proposed to be involved in the biogenesis of tER sites by binding to COPII coat components and clustering nascent-coated vesicles. Here, we show that Drosophila Sec16 (dSec16) acts instead as a tER scaffold upstream of the COPII machinery, including Sar1. We show that dSec16 is required for Sar1-GTP concentration to the tER sites where it recruits in turn the components of the COPII machinery to initiate coat assembly. Last, we show that the dSec16 domain required for its localization maps to an arginine-rich motif located in a nonconserved region. We propose a model in which dSec16 binds ER cups via its arginine-rich domain, interacts with Sar1-GTP that is generated on ER membrane by Sec12 and concentrates it in the ER cups where it initiates the formation of COPII vesicles, thus acting as a tER scaffold.     

NMR studies of the interaction between human programmed cell death 5 and human p53

Human programmed cell death 5 (PDCD5) is a protein playing a significant role in regulating both the apoptotic and paraptotic cell deaths. Resent findings show that PDCD5 is a positive regulator of Tip60 and also has a potential ability to interact with p53. Here we aim to experimentally characterize the nature of the interactions between PDCD5 and the p53 N-terminal domain (NTD) and to depict the binding mode between two proteins. The interprotein binding interfaces were determined by NMR experiments performed with PDCD5 and various fragments of p53 NTD. The binding affinity was investigated using the NMR titration experiments. Analysis revealed that the PDCD5 binding site on p53 is localized within residues 41-56 of p53 TAD2 subdomain while p53 binds preferentially to the positively charged surface region around the C-terminals of helices α3 and α5 and the N-terminal of helix α4 of PDCD5. The binding is mainly mediated through electrostatic interactions. The present data suggested a model for the interaction between PDCD5 and the p53 NTD.