The abscisic acid-related SNARE homolog NtSyr1 contributes to secretion and growth: evidence from competition with its cytosolic domain

Syntaxins and other SNARE proteins are crucial for intracellular vesicle trafficking, fusion, and secretion. Previously, we isolated the syntaxin-related protein NtSyr1 (NtSyp121) from tobacco in a screen for abscisic acid-related signaling elements, demonstrating its role in determining the abscisic acid sensitivity of K(+) and Cl(-) channels in stomatal guard cells. NtSyr1 is localized to the plasma membrane and is expressed normally throughout the plant, especially in root tissues, suggesting that it might contribute to cellular homeostasis as well as to signaling. To explore its functions in vivo further, we examined stably transformed lines of tobacco that expressed various constructs of NtSyr1, including the full-length protein and a truncated fragment, Sp2, corresponding to the cytosolic domain shown previously to be active in suppressing ion channel response to abscisic acid. Constitutively overexpressing NtSyr1 yielded uniformly high levels of protein (>10 times the wild-type levels) and was associated with a significant enhancement of root growth in seedlings but not with any obvious phenotype in mature, well-watered plants. Similar transformations with constructs encoding the Sp2 fragment of NtSyr1 showed altered leaf morphology but gave only low levels of Sp2 fragment, suggesting a strong selective pressure against plants expressing this protein. High expression of the Sp2 fragment was achieved in stable transformants under the control of a dexamethasone-inducible promoter. Sp2 expression was correlated positively with altered cellular and tissue morphology in leaves and roots and with a cessation of growth in seedlings. Overexpression of the full-length NtSyr1 protein rescued the wild-type phenotype, even in plants expressing high levels of the Sp2 fragment, supporting the idea that the Sp2 fragment interfered specifically with NtSyr1 function by competing with NtSyr1 for its binding partners. To explore NtSyr1 function in secretion, we used a green fluorescent protein (GFP)-based section assay. When a secreted GFP marker was coexpressed with Sp2 in tobacco leaves, GFP fluorescence was retained in cytosolic reticulate and punctate structures. In contrast, in plants coexpressing secreted GFP and NtSyr1 or secreted GFP alone, no GFP fluorescence accumulated within the cells. A new yellow fluorescent protein-based secretion marker was used to show that the punctate structures labeled in the presence of Sp2 colocalized with a Golgi marker. These structures were not labeled in the presence of a dominant Rab1 mutant that inhibited transport from the endoplasmic reticulum to the Golgi. We propose that NtSyr1 functions as an element in SNARE-mediated vesicle trafficking to the plasma membrane and is required for cellular growth and homeostasis.     

Changing patterns of localization of the tobacco mosaic virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection.

Tobacco mosaic virus (TMV) derivatives that encode movement protein (MP) as a fusion to the green fluorescent protein (MP:GFP) were used in combination with antibody staining to identify host cell components to which MP and replicase accumulate in cells of infected Nicotiana benthamiana leaves and in infected BY-2 protoplasts. MP:GFP and replicase colocalized to the endoplasmic reticulum (ER; especially the cortical ER) and were present in large, irregularly shaped, ER-derived structures that may represent "viral factories." The ER-derived structures required an intact cytoskeleton, and microtubules appeared to redistribute MP:GFP from these sites during late stages of infection. In leaves, MP:GFP accumulated in plasmodesmata, whereas in protoplasts, the MP:GFP was targeted to distinct, punctate sites near the plasma membrane. Treating protoplasts with cytochalasin D and brefeldin A at the time of inoculation prevented the accumulation of MP:GFP at these sites. It is proposed that the punctate sites anchor the cortical ER to plasma membrane and are related to sites at which plasmodesmata form in walled cells. Hairlike structures containing MP:GFP appeared on the surface of some of the infected protoplasts and are reminiscent of similar structures induced by other plant viruses. We present a model that postulates the role of the ER and cytoskeleton in targeting the MP and viral ribonucleoprotein from sites of virus synthesis to the plasmodesmata through which infection is spread.     

ER retention may play a role in sorting of the nuclear pore membrane protein POM121

Integral membrane proteins of the nuclear envelope (NE) are synthesized on the rough endoplasmic reticulum (ER) and following free diffusion in the continuous ER/NE membrane system are targeted to their proper destinations due to interactions of specific domains with other components of the NE. By studying the intracellular distribution and dynamics of a deletion mutant of an integral membrane protein of the nuclear pores, POM121, which lacks the pore-targeting domain, we investigated if ER retention plays a role in sorting of integral membrane proteins to the nuclear envelope. A nascent membrane protein lacking sorting determinants is believed to diffuse laterally in the continuous ER/NE lipid bilayer and expected to follow vesicular traffic to the plasma membrane. The GFP-tagged deletion mutant, POM121(1-129)-GFP, specifically distributed within the ER membrane, but was completely absent from the Golgi compartment and the plasma membrane. Experiments using fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) demonstrated that despite having very high mobility within the whole ER network (D = 0.41 +/- 0.11 micro m(2)/s) POM121(1-129)-GFP was unable to exit the ER. It was also not detected in post-ER compartments of cells incubated at 15 degrees C. Taken together, these experiments show that amino acids 1-129 of POM121 are able to retain GFP in the ER membrane and suggest that this retention occurs by a direct mechanism rather than by a retrieval mechanism. Our data suggest that ER retention might be important for sorting of POM121 to the nuclear pores.     

Dynamic regulation of caveolin-1 trafficking in the germ line and embryo of Caenorhabditis elegans

Caveolin is the major protein component required for the formation of caveolae on the plasma membrane. Here we show that trafficking of Caenorhabditis elegans caveolin-1 (CAV-1) is dynamically regulated during development of the germ line and embryo. In oocytes a CAV-1-green fluorescent protein (GFP) fusion protein is found on the plasma membrane and in large vesicles (CAV-1 bodies). After ovulation and fertilization the CAV-1 bodies fuse with the plasma membrane in a manner reminiscent of cortical granule exocytosis as described in other species. Fusion of CAV-1 bodies with the plasma membrane appears to be regulated by the advancing cell cycle, and not fertilization per se, because fusion can proceed in spe-9 fertilization mutants but is blocked by RNA interference-mediated knockdown of an anaphase-promoting complex component (EMB-27). After exocytosis, most CAV-1-GFP is rapidly endocytosed and degraded within one cell cycle. CAV-1 bodies in oocytes appear to be produced by the Golgi apparatus in an ARF-1-dependent, clathrin-independent, mechanism. Conversely endocytosis and degradation of CAV-1-GFP in embryos requires clathrin, dynamin, and RAB-5. Our results demonstrate that the distribution of CAV-1 is highly dynamic during development and provides new insights into the sorting mechanisms that regulate CAV-1 localization.     

The potato virus X TGBp2 movement protein associates with endoplasmic reticulum-derived vesicles during virus infection

The green fluorescent protein (GFP) gene was fused to the potato virus X (PVX) TGBp2 gene, inserted into either the PVX infectious clone or pRTL2 plasmids, and used to study protein subcellular targeting. In protoplasts and plants inoculated with PVX-GFP:TGBp2 or transfected with pRTL2-GFP:TGBp2, fluorescence was mainly in vesicles and the endoplasmic reticulum (ER). During late stages of virus infection, fluorescence became increasingly cytosolic and nuclear. Protoplasts transfected with PVX-GFP:TGBp2 or pRTL2-GFP:TGBp2 were treated with cycloheximide and the decline of GFP fluorescence was greater in virus-infected protoplasts than in pRTL2-GFP:TGBp2-transfected protoplasts. Thus, protein instability is enhanced in virus-infected protoplasts, which may account for the cytosolic and nuclear fluorescence during late stages of infection. Immunogold labeling and electron microscopy were used to further characterize the GFP:TGBp2-induced vesicles. Label was associated with the ER and vesicles, but not the Golgi apparatus. The TGBp2-induced vesicles appeared to be ER derived. For comparison, plasmids expressing GFP fused to TGBp3 were transfected to protoplasts, bombarded to tobacco leaves, and studied in transgenic leaves. The GFP:TGBp3 proteins were associated mainly with the ER and did not cause obvious changes in the endomembrane architecture, suggesting that the vesicles reported in GFP:TGBp2 studies were induced by the PVX TGBp2 protein. In double-labeling studies using confocal microscopy, fluorescence was associated with actin filaments, but not with Golgi vesicles. We propose a model in which reorganization of the ER and increased protein degradation is linked to plasmodesmata gating.     

Protein-binding partners of the tobacco syntaxin NtSyr1.

Syntaxins and other SNARE (soluble NSF-attachment protein receptor) complex proteins play a key role in the cellular processes of vesicle trafficking, vesicle fusion and secretion. Intriguingly, the SNARE NtSyr1 (=NtSyp121) from Nicotiana tabacum also appears to have a role in signalling evoked by the plant stress hormone abscisic acid. However, partner proteins contributing to its function(s) remain unknown. We used an affinity chromatography approach to identify proteins from tobacco leaf microsomes that directly interact with the hydrophilic (cytosolic) domains of NtSyr1 and report several interacting proteins with sensitivities to the endopeptidase activity of Clostridium botulinum neurotoxins, including one protein that was recognised by alphaAtSNAP33 antiserum, raised against the Arabidopsis SNAP25 homologue. Treatment of microsomal membrane fractions indicated a protein near 55 kDa was sensitive to proteolysis by BotN/A and BotN/E, yielding degradation products of approximately 34 and 23 kDa. Expressed and purified AtSNAP33 also bound directly to the cytosolic domain of NtSyr1 and was sensitive to proteolysis by these toxins, suggesting that NtSyr1, a tobacco homologue of AtSNAP33, and coordinate SNAREs are likely to associate as partners for function in vivo.     

A Novel Tetanus Neurotoxin-insensitive Vesicle-associated Membrane Protein in SNARE Complexes of the Apical Plasma Membrane of Epithelial Cells

The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been demonstrated that clostridial neurotoxins (NTs) proteolyze the vesicle SNAREs (v-SNAREs) vesicle-associated membrane protein (VAMP)/brevins and their partners, the target SNAREs (t-SNAREs) syntaxin 1 and SNAP25. Yet, several exocytotic events, including apical exocytosis in epithelial cells, are insensitive to numerous clostridial NTs, suggesting the presence of SNARE-independent mechanisms of exocytosis. In this study we found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs. In epithelial cells, TI-VAMP–containing vesicles were concentrated in the apical domain, and the protein was detected at the apical plasma membrane by immunogold labeling on ultrathin cryosections. Syntaxin 3 and SNAP23 were codistributed at the apical plasma membrane where they formed NEM-dependent SNARE complexes with TI-VAMP and cellubrevin. We suggest that TI-VAMP, SNAP23, and syntaxin 3 can participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specific exocytosis in clostridial NT-resistant pathways.     

Trafficking pathways of Cx49-GFP in living mammalian cells

In the present study we examined the trafficking pathways of connexin49 (Cx49) fused to green fluorescent protein (GFP) in polar and non-polar cell lines. The Cx49 gene was isolated from ovine lens by RT-PCR. Cx49 cDNA was fused to GFP and the hybrid cDNA was transfected into several cell lines. After transfection of Cx49-GFP cDNA into HeLa cells, it was shown using the double whole-cell patch-clamp technique that the expressed fusion protein was still able to form conducting gap junction channels. Synthesis, assembly, and turnover of the Cx49-GFP hybrid protein were investigated using a pulse-chase protocol. A major 78-kDa protein band corresponding to Cx49-GFP could be detected with a turnover of 16-20 h and a half-life time of 10 h. The trafficking pathways of Cx49-GFP were monitored by confocal laser microscopy. Fusion proteins were localized in subcellular compartments, including the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment, the Golgi apparatus, and the trans-Golgi network, as well as vesicles traveling towards the plasma membrane. Time-dependent sequential localization of Cx49-GFP in the ER and then the Golgi apparatus supports the notion of a slow turnover of Cx49-GFP compared to other connexins analyzed so far. Gap junction plaques resembling the usual punctuate distribution pattern could be demonstrated for COS-1 and MDCK cells. Basolateral distribution of Cx49-GFP was observed in polar MDCK cells, indicating specific sorting behavior of Cx49 in polarized cells. Together, this report describes the first characterization of biosynthesis and trafficking of lens Cx49.     

The transmembrane segment of ryanodine receptor contains an intracellular membrane retention signal for Ca(2+) release channel.

The ryanodine receptor (RyR) is a large homotetrameric protein with a hydrophobic domain at the C-terminal end that resides in the endoplasmic reticulum (ER) or sarcoplasmic reticulum membrane and forms the conduction pore of a Ca(2+) release channel. Our previous studies showed that RyR expressed in heterologous cells localized to the ER membrane. Confocal microscopic imaging indicated that the ER retention signal is likely present within the C-terminal portion of RyR, a region that contains four putative transmembrane segments. To identify the amino acid sequence responsible for ER retention of RyR, we expressed fusion proteins containing intercellular adhesion molecule (ICAM), various fragments of RyR, and green fluorescent protein (GFP) in Chinese hamster ovary and COS-7 cells. ICAM is a plasma membrane-resident glycoprotein and serves as a reporter for protein trafficking to the cell surface membrane. Imaging analyses indicated that ICAM-GFP fusion proteins with RyR sequence preceding the four transmembrane segments, ICAM-RyR-(3661-3993)-GFP, and with RyR sequence corresponding to transmembrane segments 1, 2, and 3, ICAM-RyR-(4558-4671)-GFP and ICAM-RyR-(4830-4919)-GFP, were localized to the plasma membrane; fusion proteins containing the fourth transmembrane segment of RyR, ICAM-RyR-(4913-4943)-GFP, were retained in the ER. Biochemical assay showed that ICAM-RyR-GFP fusion proteins that target to the plasma membrane are fully glycosylated, and those retained in the intracellular membrane are core-glycosylated. Together our data indicate that amino acids 4918-4943 of RyR contain the signal sequence for ER retention of the Ca(2+) release channel.     

Role of VAMP-2, VAMP-7, and VAMP-8 in constitutive exocytosis from HSY cells

We evaluated the role of VAMP-2/synaptobrevin, VAMP-7/TI-VAMP, and VAMP-8/endobrevin in exocytic pathways of HSY cells, a human parotid epithelial cell line, by coexpressing these VAMP proteins tagged with green fluorescent protein (GFP) and human growth hormone (hGH) as a secretory cargo. Exocytosis of hGH was constitutive and the fluorescent signal of hGH–GFP was observed in the Golgi area and small vesicles quickly moving throughout the cytoplasm. The cytoplasmic vesicles containing hGH overlapped well with VAMP-7-GFP, but did so scarcely with VAMP-2-GFP or VAMP-8-GFP. However, when the vesicle transport from the trans-Golgi network to the plasma membrane was arrested by incubation at 20°C for 2 h and then released by warming up to 37°C; VAMP-2-GFP and hGH were clearly colocalized together in small cytoplasmic vesicles. Neither VAMP-7-GFP nor hGH–GFP was colocalized with LAMP-1, a marker for lysosomes and late endosomes. These results suggest that (1) VAMP-2 can be one of the v-SNAREs for constitutive exocytosis; (2) VAMP-7 is involved in the constitutive exocytosis as a slow, minor v-SNARE, but not in the lysosomal transport; and (3) VAMP-8 is unlikely to be a v-SNARE for constitutive exocytosis in HSY cells.     

Internalisation of cell-penetrating peptides into tobacco protoplasts

Cells are protected from the surrounding environment by plasma membrane which is impenetrable for most hydrophilic molecules. In the last 10 years cell-penetrating peptides (CPPs) have been discovered and developed. CPPs enter mammalian cells and carry cargo molecules over the plasma membrane with a molecular weight several times their own. Known transformation methods for plant cells have relatively low efficiency and require improvement. The possibility to use CPPs as potential delivery vectors for internalisation in plant cells has been studied in the present work. We analyse and compare the uptake of the fluorescein-labeled CPPs, transportan, TP10, penetratin and pVEC in Bowes human melanoma cells and Nicotiana tabacum cultivar (cv.) SR-1 protoplasts (plant cells without cell wall). We study the internalisation efficiency of CPPs with fluorescence microscopy, spectrofluorometry and fluorescence-activated cell sorter (FACS). All methods indicate, for the first time, that these CPPs can internalise into N. tabacum cv. SR-1 protoplasts. Transportan has the highest uptake efficacy among the studied peptides, both in mammalian cells and plant protoplast. The internalisation of CPPs by plant protoplasts may open up a new effective method for transfection in plants.     

Identification of functional domains of Mid1, a stretch-activated channel component, necessary for localization to the plasma membrane and Ca2+ permeation

The Saccharomyces cerevisiae MID1 gene product (Mid1) is a stretch-activated Ca(2+)-permeable channel component required for Ca2+ influx and the maintenance of viability of cells exposed to the mating pheromone, alpha-factor. It is composed of 548-amino-acid (aa) residues with four hydrophobic segments, H1 (aa 2-22), H2 (aa 92-111), H3 (aa 337-356) and H4 (aa 366-388). It also has 16 putative N-glycosylation sites. In this study, sequentially truncated Mid1 proteins conjugated with GFP were expressed in S. cerevisiae cells. The truncated protein containing the region from H1 to H3 (Mid1(1-360)-GFP) localized normally in the plasma and endoplasmic reticulum (ER) membranes and complemented the low viability and Ca(2+)-uptake activity of the mid1 mutant, whereas Mid1(1-133)-GFP containing the region from H1 to H2 did not. Mid1(Delta3-22)-GFP lacking the H1 region failed to localize in the plasma membrane. Membrane fractionation showed that Mid1(1-22)-GFP containing only H1 localized in the plasma membrane in the presence of alpha-factor, suggesting that H1 is a signal sequence responsible for the alpha-factor-induced Mid1 delivery to the plasma membrane. The region from H1 to H3 is required for the localization of Mid1 in the plasma and ER membranes. Finally, trafficking of Mid1-GFP to the plasma membrane was dependent on the N-glycosylation of Mid1 and the transporter protein Sec12.     

Plant-specific insertions in the soybean aspartic proteinases, soyAP1 and soyAP2, perform different functions of vacuolar targeting

Most aspartic proteinases (APs) of plant origin are characterized by the presence of plant-specific insertion (PSI) in their primary structure. PSI has been reported to function as signals for both transport of AP molecules from the endoplasmic reticulum (ER) and for their targeting to the vacuole. To determine the functions of the PSIs in soyAP1 and soyAP2 identified in our previous study, we examined their subcellular localization by transient expression of a green fluorescent protein (GFP) fusion protein in the protoplasts of Arabidopsis suspension-cultured cells. Both soyAP1-GFP and soyAP2-GFP were targeted to the vacuole. To confirm the role of the PSI, we prepared PSI-deleted soyAP1 and soyAP2, and investigated their vacuolar targeting by the same method. While the former deletion mutant was always transported to the vacuole, the latter sometimes remained in the ER and was only sometimes transported to the vacuole. These observations indicated that, in the case of soyAP1, the PSI is not involved in vacuolar targeting, also suggesting that the function of the PSI differs depending on its origin.     

Multiple cytosolic and transmembrane determinants are required for the trafficking of SCAMP1 via an ER-Golgi-TGN-PM pathway

How polytopic plasma membrane (PM) proteins reach their destination in plant cells remains elusive. Using transgenic tobacco BY-2 cells, we previously showed that the rice secretory carrier membrane protein 1 (SCAMP1), an integral membrane protein with four transmembrane domains (TMDs), is localized to the PM and trans-Golgi network (TGN). Here, we study the transport pathway and sorting signals of SCAMP1 by following its transient expression in tobacco BY-2 protoplasts and show that SCAMP1 reaches the PM via an endoplasmic reticulum (ER)-Golgi-TGN-PM pathway. Loss-of-function and gain-of-function analysis of various green fluorescent protein (GFP) fusions with SCAMP1 mutations further demonstrates that: (i) the cytosolic N-terminus of SCAMP1 contains an ER export signal; (ii) the transmembrane domain 2 (TMD2) and TMD3 of SCAMP1 are essential for Golgi export; (iii) SCAMP1 TMD1 is essential for TGN-to-PM targeting; (iv) the predicted topology of SCAMP1 and its various mutants remain identical as demonstrated by protease protection assay. Therefore, both the cytosolic N-terminus and TMD sequences of SCAMP1 play integral roles in mediating its transport to the PM via an ER-Golgi-TGN pathway.     

Genesis of and trafficking to the Maurer's clefts of Plasmodium falciparum-infected erythrocytes

Malaria parasites export proteins beyond their own plasma membrane to locations in the red blood cells in which they reside. Maurer's clefts are parasite-derived structures within the host cell cytoplasm that are thought to function as a sorting compartment between the parasite and the erythrocyte membrane. However, the genesis of this compartment and the signals directing proteins to the Maurer's clefts are not known. We have generated Plasmodium falciparum-infected erythrocytes expressing green fluorescent protein (GFP) chimeras of a Maurer's cleft resident protein, the membrane-associated histidine-rich protein 1 (MAHRP1). Chimeras of full-length MAHRP1 or fragments containing part of the N-terminal domain and the transmembrane domain are successfully delivered to Maurer's clefts. Other fragments remain trapped within the parasite. Fluorescence photobleaching and time-lapse imaging techniques indicate that MAHRP1-GFP is initially trafficked to isolated subdomains in the parasitophorous vacuole membrane that appear to represent nascent Maurer's clefts. The data suggest that the Maurer's clefts bud from the parasitophorous vacuole membrane and diffuse within the erythrocyte cytoplasm before taking up residence at the cell periphery.     

In vivo FRET–FLIM reveals ER-specific increases in the ABA level upon environmental stresses

Plant hormone abscisic acid (ABA) is essential for regulating plant growth and various stress responses. ABA-mediated signaling depends on local ABA levels rather than the overall cellular ABA concentration. While cellular concentration of ABA can be detected using Förster resonance energy transfer (FRET)-based ABA probes, direct imaging of subcellular ABA levels remains unsolved. Here, we modified the previously reported ABAleon2.1 and generated a new ABA sensor, named ABAleon2.1_Tao3. Via transient expression in tobacco (Nicotiana tabacum) protoplasts, we targeted ABAleon2.1_Tao3s to the endoplasmic reticulum (ER) membrane with the ABA sensing unit facing the cytosol and the ER, respectively, through a nanobody–epitope-mediated protein interaction. Combining FRET with fluorescence lifetime imaging microscopy, ABA-triggered-specific increases in the fluorescence lifetime of the donor mTurquoise in the ABAleon2.1_Tao3 were detected in both transient assays and stably transformed Arabidopsis plants. In tobacco protoplasts, ER membrane-targeted ABAleon2.1_Tao3s showed a generally higher basal level of ABA in the ER than that in the cytosol and ER-specific alterations in the level of ABA upon environmental cues. In ABAleon2.1_Tao3-transformed Arabidopsis roots, mannitol triggered increases in cytosolic ABA in the division zone and increases in ER ABA in the elongation and maturation zone within 1 h after treatment, both of which were abolished in the bg1-2 mutant, suggesting the requirement for BG1 in osmotic stress-triggered early ABA induction in Arabidopsis roots. These data demonstrate that ABAleon2.1_Tao3s can be used to monitor ABA levels in the cytosol and the ER, providing key information on stress-induced changes in the level of ABA in different subcellular compartments.

FRET-based ABA sensors targeted to the ER membrane through a nanobody–epitope-mediated protein assembly reveal specific alterations and dynamics of ABA in the ER upon various environmental stimuli.     

On and off membrane dynamics of the endoplasmic reticulum-golgi tethering factor p115 in vivo

The mechanisms regulating membrane recruitment of the p115 tethering factor in vivo are unknown. Here, we describe cycling of p115 between membranes and cytosol and document the effects of Golgi matrix proteins, Rab1, and soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) on this process. Rapid membrane/cytosol exchange is shown by swift (t1/2 approximately 20 s) loss of Golgi-localized p115-green fluorescent protein (GFP) after repeated photobleaching of cell periphery and rapid (t1/2 approximately 13 s) fluorescence recovery after photobleaching Golgi-localized p115-GFP. p115 mutant missing the GM130/giantin binding site exhibits analogous fluorescence recovery after photobleaching (FRAP) (t1/2 approximately 13 s), suggesting that GM130 and giantin are not major determinants of p115 membrane dynamics. In contrast, p115-GFP exchanges more rapidly (t1/2 approximately 8 s) in cells expressing the inactive Rab1/N121I mutant, indicating that p115 cycling is influenced by Rab1. p115-GFP dynamics is also influenced by the assembly status of SNAREs. In cells expressing an ATPase-deficient NSF/E329Q mutant that inhibits SNARE complex disassembly, the cycling kinetics of p115-GFP are significantly slower (t1/2 approximately 21 s). In contrast, in cells incubated at reduced temperature (10 degrees C) that inhibits vesicular traffic, the cycling kinetics of p115-GFP are faster (t1/2 approximately 7 s). These data suggest that p115-binding sites on the membrane are provided by unassembled SNAREs. In agreement, biochemical studies show increased p115 recruitment to membranes in the presence of NSF and alpha-SNAP. Our data support a model in which recruitment of tethers is directly regulated by the assembly status of SNAREs.     

Syntaxin isoform specificity in the regulation of renal H+-ATPase exocytosis

Intercalated and inner medullary collecting duct (IMCD) cells of the kidney mediate the transport of H+ by a plasma membrane H+-ATPase. The rate of H+ transport in these cells is regulated by exocytic insertion of H+-ATPase-laden vesicles into the apical membrane. We have shown that the exocytic insertion of proton pumps (H+-ATPase) into the apical membrane of rat IMCD cells, in culture, involves SNARE proteins (syntaxin (synt), SNAP-23, and VAMP). The membrane fusion complex observed in IMCD cells with the induction of proton pump exocytosis not only included these SNAREs but also the H+-ATPase. Based on these observations, we suggested that the targeting of these vesicles to the apical membrane is mediated by an interaction between the H+-ATPase and a specific t-SNARE. To evaluate this hypothesis, we utilized a "pull-down" assay in which we identified, by Western analysis, the proteins in a rat kidney medullary homogenate that complexed with glutathione S-transferase (GST) fusion syntaxin isoforms attached to Sepharose 4B-glutathione beads. The syntaxin isoforms employed were 1A, 1B, 2, 4, 5, and also 1A that was truncated to exclude the H3 SNARE binding domain (synt-1ADeltaH3). All full-length syntaxin isoforms formed complexes with SNAP-23 and VAMP. Neither GST nor synt-1ADeltaH3 formed complexes with these SNAREs. H+-ATPase (subunits E, a, and c) bound to syntaxin-1A and to a lesser extent to synt-1B but not to synt-1ADeltaH3 or synt-2, -4, and -5. In cultured IMCD cells transfected to express syntaxin truncated for the membrane binding domain (synt-DeltaC), expression of synt-1ADeltaC, but not synt-4DeltaC, inhibited H+-ATPase exocytosis. In conclusion, because all full-length syntaxins examined bound VAMP-2 and SNAP-23, but only non-H3-truncated syntaxin-1 bound H+-ATPase, and synt-1ADeltaC expression by intact IMCD cells inhibited H+-ATPase exocytosis, it is likely that the H+-ATPase binds directly to the H3 domain of syntaxin-1 and not through VAMP-2 or SNAP-23. Interaction between the syntaxin-1A and H+-ATPase is important in the targeted exocytosis of the proton pump to the apical membrane of intercalated cells.     

Aspects of plant plasmalemma charging induced by external electric field pulses

The charging of the plasmalemma is a necessary condition for permeabilization of the plasma membrane (electroporation) in response to external electric field exposure. Common theories explain this permeabilization by formation of pores in the lipid bilayer. Using pulsed laser fluorescence microscopy, we measured the charging process of the membrane during the application of an external electric field with a temporal resolution of 5 ns. Visualization of the charging process of protoplasts plasma membrane (Nicotiana tabacum Bright Yellow 2) was achieved by staining of the plasma membrane with the voltage-sensitive fluorescent dye ANNINE-6. Measurements on membranes exhibiting negligible membrane permeabilization confirm the sine-shaped azimuthal distribution of the membrane voltage predicted by the relation of Cole. At higher membrane voltages, enhanced pore formation allows for the exchange of charge carriers, leading to deviations from the sine-shaped curve progression, i.e., a saturation of the membrane voltage at membrane segments facing the electrodes. Additionally, measurements on protoplasts exposed to multiple successive pulses indicate that the recovery of the membrane seems to be a fast process, occurring within seconds after termination of the external electric field pulse.     

Flow sorting and culture of protoplasts: Conditions for high-frequency recovery,growth and morphogenesis from sorted protoplasts of suspension cultures of nicotiana

Protoplasts were prepared from suspension cultures of Nicotiana tabacum cv Wisconsin 38 that had been prelabeled with FITC. The protoplasts were subjected to flow sorting based on fluorescence content using a Coulter EPICS V Flow Cytometer — Cell Sorter. Conditions were established that allowed the recovery after sorting of approximately 30% of the initial protoplasts in a viable state. These were subsequently regenerated into calli that underwent shoot morphogenesis.Abbreviations FITC Fluorescein isothiocyanate - FDA fluorescein diacetate