Siew Heng Wong

SNX3 regulates endosomal function through its PX−domain−mediated interaction with PtdIns(3)P

The sorting nexin (SNX) protein family is implicated in regulating membrane traffic, but the mechanism is still unknown. We show that SNX3 is associated with the early endosome through a novel motif (PX domain) capable of interaction with phosphatidylinositol-3-phosphate (PtdIns(3)P). Overexpression of SNX3 alters endosomal morphology and delays transport to the lysosome. Transport from the early to the recycling endosome is affected upon microinjection of SNX3 antibodies. Our results highlight a novel mechanism by which SNX proteins regulate traffic and uncover a novel class of effectors for PtdIns(3)P.

Overexpression of E2F-1 in rat embryo fibroblasts leads to neoplastic transformation.

The transcription factor E2F has been implicated in controlling the activation of multiple genes associated with cell proliferation. E2F‐1, which is a component of E2F, can promote oncogenesis when transfected into REF cells. The transformation caused by E2F‐1 correlates with constitutive overexpression of the transgene, increased transcription of E2F‐dependent genes and the enhancement of two E2F DNA binding complexes containing the retinoblastoma susceptibility gene product (Rb) and E2F‐1. The oncogenic potential of E2F‐1 is dependent on functional DNA binding and transactivation domains but does not require the ability to interact directly with Rb. These findings provide the first direct evidence that sustained unregulated expression of E2F‐1 can lead to the loss of cell proliferation control and that E2F‐1 is a key component in cell cycle control.

GS28, a 28-Kilodalton Golgi SNARE That Participates in ER-Golgi Transport

Little is known about the integral membrane proteins that participate in the early secretory pathway of mammalian cells. The complementary DNA encoding a 28-kilodalton protein (p28) of the cis-Golgi was cloned and sequenced. The protein was predicted to contain a central coiled-coil domain with a carboxyl-terminal membrane anchor. An in vitro assay for endoplasmic reticulum-Golgi transport was used to show that p28 participates in the docking and fusion stage of this transport event. Biochemical studies established that p28 is a core component of the Golgi SNAP receptor (SNARE) complex.

Syntaxin 7, a Novel Syntaxin Member Associated with the Early Endosomal Compartment

Members of the syntaxin family are key molecules involved in diverse vesicle docking/fusion events. We report here the molecular, biochemical, and cell biological characterizations of a novel member (syntaxin 7) of the syntaxin family. Syntaxin 7 is structurally related to all known syntaxins. Within a 79-residue region preceding the C-terminal hydrophobic tail, syntaxin 7 is 35, 34, 34, 34, 25, and 19% identical to syntaxins 1, 2, 3, 4, 5, and 6, respectively. Northern blot analysis showed that syntaxin 7 is widely expressed. Indirect immunofluorescence microscopy revealed that syntaxin 7 is primarily associated with the early endosome. In vitro binding assays established that syntaxin 7 in membrane extracts interacts with immobilized recombinant α-solubleN-ethylmaleimide-sensitive factor attachment proteins fused to glutathione S-transferase. Our results highlight the general importance of members of the syntaxin family in protein trafficking and provide new avenues for future functional and mechanistic studies of this first endosomal syntaxin as well as the endocytotic pathway.

A SNARE involved in protein transport through the Golgi apparatus

In eukaryotic cells, the Golgi apparatus receives newly synthesized proteins from the endoplasmic reticulum (ER) and delivers them after covalent modification to their destination in the cell. These proteins move from the inside (cis) face to the plasma-membrane side (trans) of the Golgi, through a stack of cisternae, towards the trans-Golgi network (TGN), but very little is known about how proteins are moved through the Golgi compartments. In a model known as the maturation model, no special transport process was considered necessary, with protein movement along the Golgi being achieved by maturation of the cisternae. Alternatively, proteins could be transported by vesicles or membrane tubules,. Although little is known about membrane-tubule-mediated transport,, the molecular mechanism for vesicle-mediated transport is quite well understood, occurring through docking of SNAREs on the vesicle with those on the target membrane. We have now identified a protein of relative molecular mass 27K which is associated with the Golgi apparatus. The cytoplasmic domain of this protein or antibodies raised against it quantitatively inhibit transport in vitro from the ER to the trans-Golgi/TGN, acting at a stage between the cis/medial- and the trans-Golgi/TGN. This protein, which behaves like a SNARE and has been named GS27 (for Golgi SNARE of 27K), is identical to membrin, a protein implicated earlier in ER-to-Golgi transport. Our results suggest that protein movement from medial- to the trans-Golgi/TGN depends on SNARE-mediated vesicular transport.

A 29-Kilodalton Golgi Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (Vti1-rp2) Implicated in Protein Trafficking in the Secretory Pathway*

Expressed sequence tags coding for a potential SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) were revealed during data base searches. The deduced amino acid sequence of the complete coding region predicts a 217-residue protein with a COOH-terminal hydrophobic membrane anchor. Affinity-purified antibodies raised against the cytoplasmic region of this protein specifically detect a 29-kilodalton integral membrane protein enriched in the Golgi membrane. Indirect immunofluorescence microscopy reveals that this protein is mainly associated with the Golgi apparatus. When detergent extracts of the Golgi membrane are incubated with immobilized glutathione S-transferase α soluble N-ethylmaleimide-sensitive factor attachment protein (GST-α-SNAP), this protein was specifically retained. This protein has been independently identified and termed Vti1-rp2, and it is homologous to Vti1p, a yeast Golgi SNARE. We further show that Vti1-rp2 can be qualitatively coimmunoprecipitated with Golgi syntaxin 5 and syntaxin 6, suggesting that Vti1-rp2 exists in at least two distinct Golgi SNARE complexes. In cells microinjected with antibodies against Vti1-rp2, transport of the envelope protein (G-protein) of vesicular stomatitis virus from the endoplasmic reticulum to the plasma membrane was specifically arrested at the Golgi apparatus, providing further evidence for functional importance of Vti1-rp2 in protein trafficking in the secretory pathway.

GS15, a 15-kilodalton Golgi soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) homologous to rbet1

Bet1p plays an essential role in vesicular transport from the endoplasmic reticulum (ER) to the Golgi in yeast, and it functions as a vesicle solubleN-ethylmaleimide-sensitive factor protein receptor (v-SNARE). A mammalian protein related to Bet1p has been reported previously and was referred to as rbet1. We have now identified a new mammalian protein that is homologous to rbet1 (28% amino acid identity). mRNA for this rbet1 homologue is widely expressed in rat tissues. Affinity-purified polyclonal antibodies raised against recombinant protein specifically recognized a 15-kilodalton integral membrane protein highly enriched in Golgi membranes. Indirect immunofluorescence microscopy revealed that this protein is specifically associated with the Golgi apparatus in diverse cell types. Biochemical characterization established that this protein behaves like a SNARE and was named GS15 (Golgi SNARE with a size of 15 kilodaltons). These properties raise the possibility that GS15 is a novel SNARE mediating a yet to be defined transport event associated with the Golgi apparatus.

Mutually exclusive interactions of EHD1 with GS32 and Syndapin II

GS32/SNAP-29 is a SNAP-25-like SNARE and has been shown to interact with syntaxin 6. Using immobilized recombinant GS32, we have recovered EHD1 as a major GS32-interacting protein from total HeLa cell extracts. This interaction is mediated by the EH domain of EHD1 and the N-terminal NPF-containing 17-residue region of GS32. Co-immunoprecipitation suggests that GS32 could also interact with EHD1 in intact cells. When immobilized GST-EHD1 was used to fish out interacting proteins from total brain extracts, syndapin II was identified as a major interacting partner. Similar to the GS32-EHD1 interaction, syndapin II also interacts with the EH domain of EHD1 via its NPF repeat region. Interaction of endogenous EHD1 and syndapin II was also established by co-immunoprecipitation. Furthermore, interaction of GS32 and syndapin II with EHD1 was shown to be mutually exclusive, suggesting that EHD1 may regulate/participate in the functional pathways of both GS32 and syndapin II in a mutual exclusive manner. Opposing roles of GS32 and syndapin II in regulating the surface level of transferrin receptor (TfR) were observed.

Transmembrane topology of the mammalian KDEL receptor.

The mammalian KDEL receptor is an integral membrane protein with seven hydrophobic regions. Fusion proteins comprising a 37-kDa N-glycosylation reporter fused downstream of amino-terminal fragments of the KDEL receptor with varying numbers of hydrophobic regions were synthesized in an in vitro translation system containing canine pancreatic microsomes. The luminal or cytosolic orientation of the reporter, and hence of the hydrophilic region to which it is fused, was inferred from the presence or absence of glycosylation, which occurs only in the lumen of the microsomes. The cytosolic orientation of the N and C termini was also confirmed immunocytochemically. Our results suggest that the KDEL receptor is inserted into the membrane with only six transmembrane domains and that both the amino and carboxy termini are located in the cytoplasm.

Vesicle-Associated Membrane Protein-8/Endobrevin Negatively Regulates Phagocytosis of Bacteria in Dendritic Cells

Phagocytosis is a specialized mechanism used by mammalian cells, particularly the cells of the immune system, such as dendritic cells (DC) and macrophages, to protect the host against infection. The process involves a complex cascade of pathways, from the ligation of surface receptors of phagocytes with components of the microorganism’s surface, formation of phagosomes and subsequently phagolysosomes, to the eventual presentation of foreign Ags. Vesicle-associated membrane protein (VAMP)-8/endobrevin has been shown previously to function in the endocytic pathways. Our results showed that VAMP-8 colocalized with lysosome-associated membrane protein-2, and a significant amount of VAMP-8 was recruited to the phagosomes during bacterial ingestion. However, overexpression of VAMP-8 significantly inhibited phagocytosis in DC. We also found that the phagocytic activity of VAMP-8−/− DC was significantly higher than wild-type VAMP-8+/+ DC, thus further confirming that VAMP-8 negatively regulates phagocytosis in immature DC.