The exomer cargo adaptor structure reveals a novel GTPase‐binding domain

Cargo adaptors control intracellular trafficking of transmembrane proteins by sorting them into membrane transport carriers. The COPI, COPII, and clathrin cargo adaptors are structurally well characterized, but other cargo adaptors remain poorly understood. Exomer is a specialized cargo adaptor that sorts specific proteins into trans‐Golgi network (TGN)‐derived vesicles in response to cellular signals. Exomer is recruited to the TGN by the Arf1 GTPase, a universally conserved trafficking regulator. Here, we report the crystal structure of a tetrameric exomer complex composed of two copies each of the Chs5 and Chs6 subunits. The structure reveals the FN3 and BRCT domains of Chs5, which together we refer to as the FBE domain (F N3–B RCT of e xomer), project from the exomer core complex. The overall architecture of the FBE domain is reminiscent of the appendage domains of other cargo adaptors, although it exhibits a distinct topology. In contrast to appendage domains, which bind accessory factors, we show that the primary role of the FBE domain is to bind Arf1 for recruitment of exomer to membranes.     

Cargo selection in vesicular transport: the making and breaking of a coat

Intracellular traffic is mediated by vesicular/tubular carriers. The carriers are formed by the activity of cytosolic coat proteins that are recruited to their target membranes and deform these membranes into buds and vesicles. Specific interactions between recruited coat subunits and short peptide sequences (transport motifs) on cargo proteins direct the incorporation of cargo into budded vesicles. Here, we focus on cargo selection reactions mediated by COPII and AP-2/clathrin vesicle coat complexes to explore common mechanisms by which coat assembly support localized and selective cargo sorting. Recent findings suggest that multiple, low-affinity interactions are employed in a cooperative manner to support coat assembly and enable cargo recognition. Thus low-binding affinities between coat subunits and transport motifs are transiently transformed into high-avidity, multivalent and selective interactions at vesicle bud sites. The temporal and regulated nature of the interactions provide the key to cargo selection.     

Lipid-anchored Synaptobrevin Provides Little or No Support for Exocytosis or Liposome Fusion

SNARE proteins catalyze many forms of biological membrane fusion, including Ca²⁺-triggered exocytosis. Although fusion mediated by SNAREs generally involves proteins anchored to each fusing membrane by a transmembrane domain (TMD), the role of TMDs remains unclear, and previous studies diverge on whether SNAREs can drive fusion without a TMD. This issue is important because it relates to the question of the structure and composition of the initial fusion pore, as well as the question of whether SNAREs mediate fusion solely by creating close proximity between two membranes versus a more active role in transmitting force to the membrane to deform and reorganize lipid bilayer structure. To test the role of membrane attachment, we generated four variants of the synaptic v-SNARE synaptobrevin-2 (syb2) anchored to the membrane by lipid instead of protein. These constructs were tested for functional efficacy in three different systems as follows: Ca²⁺-triggered dense core vesicle exocytosis, spontaneous synaptic vesicle exocytosis, and Ca²⁺-synaptotagmin-enhanced SNARE-mediated liposome fusion. Lipid-anchoring motifs harboring one or two lipid acylation sites completely failed to support fusion in any of these assays. Only the lipid-anchoring motif from cysteine string protein-α, which harbors many lipid acylation sites, provided support for fusion but at levels well below that achieved with wild type syb2. Thus, lipid-anchored syb2 provides little or no support for exocytosis, and anchoring syb2 to a membrane by a TMD greatly improves its function. The low activity seen with syb2-cysteine string protein-α may reflect a slower alternative mode of SNARE-mediated membrane fusion.     

NbRABG3f,a member of Rab GTPase,is involved in Bamboo mosaic virus infection in Nicotiana benthamiana

The screening of differentially expressed genes in plants after pathogen infection can uncover the potential host factors required for the pathogens. In this study, an up‐regulated gene was identified and cloned from Nicotiana benthamiana plants after Bamboo mosaic virus (BaMV) inoculation. The up‐regulated gene was identified as a member of the Rab small guanosine triphosphatase (GTPase) family, and was designated as NbRABG3f according to its in silico translated product with high identity to that of RABG3f of tomato. Knocking down the expression of NbRABG3f using a virus‐induced gene silencing technique in a protoplast inoculation assay significantly reduced the accumulation of BaMV. A transiently expressed NbRABG3f protein in N. benthamiana plants followed by BaMV inoculation enhanced the accumulation of BaMV to approximately 150%. Mutants that had the catalytic site mutation (NbRABG3f/T22N) or had lost their membrane‐targeting capability (NbRABG3f/ΔC3) failed to facilitate the accumulation of BaMV in plants. Because the Rab GTPase is responsible for vesicle trafficking between organelles, a mutant with a fixed guanosine diphosphate form was used to identify the donor compartment. The use of green fluorescent protein (GFP) fusion revealed that GFP‐NbRABG3f/T22N clearly co‐localized with the Golgi marker. In conclusion, BaMV may use NbRABG3f to form vesicles derived from the Golgi membrane for intracellular trafficking to deliver unidentified factors to its replication site; thus, both GTPase activity and membrane‐targeting ability are crucial for BaMV accumulation at the cell level.     

The integration of autophagy and cellular trafficking pathways via RAB GAPs

Macroautophagy is a conserved degradative pathway in which a double-membrane compartment sequesters cytoplasmic cargo and delivers the contents to lysosomes for degradation. Efficient formation and maturation of autophagic vesicles, so-called phagophores that are precursors to autophagosomes, and their subsequent trafficking to lysosomes relies on the activity of small RAB GTPases, which are essential factors of cellular vesicle transport systems. The activity of RAB GTPases is coordinated by upstream factors, which include guanine nucleotide exchange factors (RAB GEFs) and RAB GTPase activating proteins (RAB GAPs). A role in macroautophagy regulation for different TRE2-BUB2-CDC16 (TBC) domain-containing RAB GAPs has been established. Recently, however, a positive modulation of macroautophagy has also been demonstrated for the TBC domain-free RAB3GAP1/2, adding to the family of RAB GAPs that coordinate macroautophagy and additional cellular trafficking pathways.     

Differential dynamics of alpha 5 integrin, paxillin, and alpha-actinin during formation and disassembly of adhesions in migrating cells.

To investigate the mechanisms by which adhesions form and disperse in migrating cells, we expressed alpha 5 integrin, alpha-actinin, and paxillin as green fluorescent protein (GFP) fusions. All localized with their endogenous counterparts and did not perturb migration when expressed at moderate levels. alpha 5-GFP also rescued the adhesive defects in CHO B2 cells, which are alpha 5 integrin deficient. In ruffling cells, alpha 5-GFP and alpha-actinin--GFP localized prominently at the leading edge in membrane protrusions. Of the three GFP fusion proteins that we examined, paxillin was the first component to appear visibly organized in protrusive regions of the cell. When a new protrusion formed, the paxillin appeared to remodel from older to newer adhesions at the leading edge. alpha-Actinin subsequently entered adhesions, which translocated toward the cell center, and inhibited paxillin turnover. The new adhesions formed from small foci of alpha-actinin--GFP and paxillin-GFP, which grew in size. Subsequently, alpha 5 integrin entered the adhesions to form visible complexes, which served to stabilize the adhesions. alpha 5-GFP also resided in endocytic vesicles that emanated from the leading edge of protrusions. Integrin vesicles at the cell rear moved toward the cell body. As cells migrated, alpha 5 vesicles also moved from a perinuclear region to the base of the lamellipodium. The alpha 5 vesicles colocalized with transferrin receptor and FM 4-64 dye. After adhesions broke down in the rear, alpha 5-GFP was found in fibrous structures behind the cell, whereas alpha-actinin--GFP and paxillin-GFP moved up the lateral edge of retracting cells as organized structures and then dissipated.     

YKT6 is a core constituent of membrane fusion machineries at the Arabidopsis trans-Golgi network

SNARE complex formation is essential for membrane fusion in exocytotic and vacuolar trafficking pathways. Vesicle-associated (v-) SNARE associates with a target membrane (t-) SNARE to form a SNARE complex bridging two membranes, which may facilitate membrane fusion. The Arabidopsis genome encodes a large number of predicted SNARE proteins that might function primarily as fusogens for vesicle transport in endomembrane systems. The SNAREs SYP41, SYP61 and VTI12 reside in the trans-Golgi network and have been proposed to function together in vesicle fusion with this organelle. Here, we use a liposome fusion assay to demonstrate that VTI12 and either SYP41 or SYP61, but not both, are required for membrane fusion. This indicates that SYP41 and SYP61 are likely to function in independent vesicle fusion reactions in Arabidopsis. In addition, we have identified two new functionally interchangeable components, YKT61 and YKT62, that show sequence similarity to the multifunctional yeast SNARE YKT6. Both YKT61 and YKT62 interact with SYP41 and are essential for membrane fusion mediated by either SYP41 or SYP61. These results therefore define the core constituents required for membrane fusion at the Arabidopsis trans-Golgi network.     

细胞内膜泡运输中拴系因子的功能

膜泡运输是不同细胞器间进行物质传递的基本方式,分为4个重要步骤：囊泡的出芽、转运、拴系和融合。在此过程中,有许多相关因子参与调控,如包被蛋白、Rab蛋白、拴系因子、SM蛋白和SNARE等。拴系因子在运输囊泡和靶位膜发生接触的最初阶段起重要调控作用,多数拴系因子形成大的多亚基复合体发挥功能。目前,关于拴系因子的功能已经有了一定的了解,在此,我们对酵母、哺乳动物以及植物细胞中的已知拴系因子的特点和功能进行了概述。