Opposite Beet Cyst Nematode Infection Phenotypes of Transgenic Arabidopsis Between Overexpressing GmSNAP18 and AtSNAP2 and Between Overexpressing GmSHMT08 and AtSHMT4.

The rhg1-a GmSNAP18 (an α-SNAP) and Rhg4 GmSHMT08 are two major cloned genes conferring soybean cyst nematode resistance in Peking-type soybeans, but the application of α-SNAPs and SHMTs in cyst nematode management remains elusive. In this study, GmSNAP18 and GmSHMT08, together with their orthologs in Arabidopsis, AtSNAP2 (an α-SNAP) and AtSHMT4, were individually transformed into Arabidopsis Col-0 to generate the transgenic lines, and the growth of transgenic plants, beet cyst nematode (BCN) infection phenotypes, and AtSNAP2, AtSHMT4, and AtPR1 expression patterns were analyzed using Arabidopsis-BCN compatible interaction system, in addition with protein-protein interaction assay. Pulldown and BiFC assays revealed that GmSNAP18 and GmSHMT08 interacted with AtSHMT4 and AtSNAP2, respectively. Plant root growth was not impacted by overexpression of GmSNAP18 and AtSNAP2. However, overexpression of GmSHMT08 and AtSHMT4 both increased plant height, additionally, overexpression of GmSHMT08 decreased rosette leaf size. Overexpression of GmSNAP18 and GmSHMT08 both suppressed AtPR1 expression and significantly enhanced BCN susceptibility, while overexpression of AtSNAP2 and AtSHMT4 both substantially boosted AtPR1 expression and remarkably enhanced BCN resistance, in transgenic Arabidopsis. Overexpression of GmSNAP18 reduced, while overexpression of AtSNAP2 unaltered AtSHMT4 expression. Overexpression of GmSHMT08 and AtSHMT4 both suppressed AtSNAP2 expression in transgenic Arabidopsis. Thus, different expression patterns of AtPR1 and AtSHMT4 are likely associated with opposite BCN infection phenotypes of Arabidopsis between overexpressing GmSNAP18 and AtSNAP2, and between overexpressing GmSHMT08 and AtSHMT4; and boosted AtPR1 expression are required for enhanced BCN resistance in Arabidopsis. All these results establish a basis for extension of α-SNAPs and SHMTs in cyst nematode management.

Vitamin E prevents hyperoxia-induced loss of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins in the rat neuronal cytoplasm.

This study examines the ability of vitamin E to inhibit hyperoxia-induced loss of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins in the neuronal cytoplasm. Here, the effects of vitamin E on hyperoxia-induced changes in the expressions of N-ethylmaleimide-sensitive factor (NSF) and soluble NSF-attachment protein α (α-SNAP) in the rat brain were analyzed. When rats were subjected to hyperoxia, the expression of both SNARE proteins was markedly decreased compared to normal rats. Vitamin E significantly inhibited the decrease in the expression of NSF in rats subjected to hyperoxia. Rats showed the tendency to improve the loss of α-SNAP by vitamin E-supplementation, although it was not statistically significant. On the other hand, vitamin E deficient rats showed marked loss of these proteins in the brain in the absence of oxidative stress. These results suggest that hyperoxia induces a loss of SNARE proteins, which are involved in membrane docking between synaptic vesicles and pre-synaptic membranes, and that vitamin E prevents the oxidative damage of SNARE proteins. Consequently, it is implied that vitamin E inhibits impaired neurotransmission caused by oxidative stress through the prevention of oxidative damage to SNARE proteins by probably its antioxidant effect.

GEC1 interacts with the kappa opioid receptor and enhances expression of the receptor.

We identified a truncated form (38-117) of GEC1 that interacts with the C-tail of the human kappa opioid receptor (hKOR) by yeast two-hybrid screening. GEC1-(38-117) did not interact with the C-tail of the mu or delta opioid receptors. GEC1, a 117-amino acid protein (Pellerin, I., Vuillermoz, C., Jouvenot, M., Ordener, C., Royez, M., and Adessi, G. L. (1993) Mol. Cell Endocrinol. 90, R17-R21), is highly homologous to GABARAP, GATE-16, and Apg8/aut7, all members of the microtubule associated protein (MAP) family. In pull-down assays, GST-GEC1 interacted directly with the hKOR C-tail, full-length hKOR, and tubulin. When expressed in Chinese hamster ovary (CHO) cells, GEC1 co-immunoprecipitated with FLAG-hKOR. Expression of GEC1 greatly increased total and cell-surface KOR but not mu or delta opioid receptors. GEC1 expression slightly reduced U50,488H-promoted down-regulation, without affecting ligand binding affinity, receptor-G protein coupling, or U50,488H-induced desensitization and internalization. HA-GEC1 expressed in CHO cells was localized in the Golgi apparatus and endoplasmic reticulum (ER). When cells were pulsed with [35S]Met/Cys, GEC1 expression enhanced the level of the mature form (55-kDa band) of FLAG-hKOR at 4, 8, and 22 h after pulse without affecting the precursors (39- and 45-kDa bands), indicating that GEC1 facilitates trafficking of FLAG-hKOR from the ER/Golgi to plasma membranes. GEC1 interacted with N-ethylmaleimide-sensitive factor (NSF) in pull-down assays and co-immunoprecipitated with NSF in rat brain extracts. The interaction with NSF may contribute to GEC1 effects. This is the first report on biological functions of GEC1 and the first demonstration that a GPCR interacts with a protein of the MAP family. The interaction is important for trafficking of the receptor in the biosynthesis pathway.

Mammalian Bet3 functions as a cytosolic factor participating in transport from the ER to the Golgi apparatus.

The TRAPP complex identified in yeast regulates vesicular transport in the early secretory pathway. Although some components of the TRAPP complex are structurally conserved in mammalian cells, the function of the mammalian components has not been examined. We describe our biochemical and functional analysis of mammalian Bet3, the most conserved component of the TRAPP complex. Bet3 mRNA is ubiquitously expressed in all tissues. Antibodies raised against recombinant Bet3 specifically recognize a protein of 22 kDa. In contrast to yeast Bet3p, the majority of Bet3 is present in the cytosol. To investigate the possible involvement of Bet3 in transport events in mammalian cells, we utilized a semi-intact cell system that reconstitutes the transport of the envelope glycoprotein of vesicular stomatitis virus (VSV-G) from the ER to the Golgi apparatus. In this system, antibodies against Bet3 inhibit transport in a dose-dependent manner, and cytosol that is immunodepleted of Bet3 is also defective in this transport. This defect can be rescued by supplementing the Bet3-depleted cytosol with recombinant GST-Bet3. We also show that Bet3 acts after COPII but before Rab1, alpha-SNAP and the EGTA-sensitive stage during ER-Golgi transport. Gel filtration analysis demonstrates that Bet3 exists in two distinct pools in the cytosol, the high-molecular-weight pool may represent the TRAPP complex, whereas the other probably represents the monomeric Bet3.

Antisense suppression of a potato alpha-SNAP homologue leads to alterations in cellular development and assimilate distribution.

Using the cDNA-AFLP method, we have isolated a transcript-derived fragment (TDF) which shows a differential expression pattern during tuber organogenesis of Solanum tuberosum L. The TDF was used to isolate a cDNA clone carrying a 1.5 kb insert and potentially coding for a 32.5 kDa peptide which, by homology, represents a potato homologue of an alpha-snap gene and has been designated Stsnap. Northern analysis showed that the Stsnap gene is expressed in actively dividing tissues throughout the potato plant. Analysis of genomic DNA from potato revealed that the Stsnap gene is likely to be a single-copy gene. The expression of antisense Stsnap cDNA under the control of the CaMV 35S promoter results in plants with an altered morphology such as curled leaves. Several of these transgenic lines also display cellular and developmental abnormalities with distinct changes in assimilate transport including accumulation of starch and soluble sugars in source leaves. We argue that these findings are consistent with the hypothetical function of the StSNAP gene product in vesicle targeting and fusion during plant development.

Alpha-SNAP but not gamma-SNAP is required for ER-Golgi transport after vesicle budding and the Rab1-requiring step but before the EGTA-sensitive step.

N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs) have been implicated in diverse vesicular transport events; yet their exact role and site of action remain to be established. Using an established in vitro system, we show that antibodies against alpha-SNAP inhibit vesicle transport from the ER to the cis-Golgi and that recombinant alpha-SNAP enhances/stimulates the process. Cytosol immunodepleted of alpha-SNAP does not support normal transport unless supplemented with recombinant alpha-SNAP but not gamma-SNAP. In marked contrast, cytosol immunodepleted of gamma-SNAP supports ER-Golgi transport to the normal level. Neither antibodies against gamma-SNAP nor recombinant gamma-SNAP have any effect on ER-Golgi transport. These results clearly establish an essential role for alpha-SNAP but not gamma-SNAP in ER-Golgi transport. When the transport assay is performed with cytosol immunodepleted of alpha-SNAP, followed by incubation with cytosol immunodepleted of a COPII subunit, normal transport is achieved. In marked contrast, no transport is detected when the assay is first performed with cytosol depleted of the COPII subunit followed by alpha-SNAP-depleted cytosol, suggesting that alpha-SNAP is required after a step that requires COPII (the budding step). In combination with cytosol immunodepleted of Rab1, it is seen that alpha-SNAP is required after a Rab1-requiring step. It has been shown previously that EGTA blocks ER-Golgi transport at a step after vesicle docking but before fusion and we show here that alpha-SNAP acts before the step that is blocked by EGTA. Our results suggest that alpha-SNAP may be involved in the pre-docking or docking but not the fusion process.

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.