A putative plant aminophospholipid flippase, the Arabidopsis P4 ATPase ALA1, localizes to the plasma membrane following association with a β-subunit

Plasma membranes in eukaryotic cells display asymmetric lipid distributions with aminophospholipids concentrated in the inner leaflet and sphingolipids in the outer leaflet. This unequal distribution of lipids between leaflets is, amongst several proposed functions, hypothesized to be a prerequisite for endocytosis. P4 ATPases, belonging to the P-type ATPase superfamily of pumps, are involved in establishing lipid asymmetry across plasma membranes, but P4 ATPases have not been identified in plant plasma membranes. Here we report that the plant P4 ATPase ALA1, which previously has been connected with cold tolerance of Arabidopsis thaliana, is targeted to the plasma membrane and does so following association in the endoplasmic reticulum with an ALIS protein β-subunit.     

Growth and metabolic control of lipid signalling at the Golgi

PtdIns4P is a key regulator of the secretory pathway and plays an essential role in trafficking from the Golgi. Our recent work demonstrated that spatial control of PtdIns4P at the ER (endoplasmic reticulum) and Golgi co-ordinates secretion with cell growth. The central elements of this regulation are specific phosphoinositide 4-kinases and the phosphoinositide phosphatase Sac1. Growth-dependent translocation of Sac1 between the ER and Golgi modulates the levels of PtdIns4P and anterograde traffic at the Golgi. In yeast, this mechanism is largely dependent on the availability of glucose, but our recent results in mammalian cells suggest that Sac1 phosphatases play evolutionarily conserved roles in the growth control of secretion. Sac1 lipid phosphatase plays also an essential role in the spatial control of PtdIns4P at the Golgi complex. A restricted pool of PtdIns4P at the TGN (trans-Golgi network) is required for Golgi integrity and for proper lipid and protein sorting. In mammalian cells, the stress-activated MAPK (mitogen-activated protein kinase) p38 appears to play a critical role in transmitting nutrient signals to the phosphoinositide signalling machinery at the ER and Golgi. These results suggest that temporal and spatial integration of metabolic and lipid signalling networks at the Golgi is required for controlling the secretory pathway.     

The Arabidopsis Rab GTPase RabA4b localizes to the tips of growing root hair cells

Spatial and temporal control of cell wall deposition plays a unique and critical role during growth and development in plants. To characterize membrane trafficking pathways involved in these processes, we have examined the function of a plant Rab GTPase, RabA4b, during polarized expansion in developing root hair cells. Whereas a small fraction of RabA4b cofractionated with Golgi membrane marker proteins, the majority of this protein labeled a unique membrane compartment that did not cofractionate with the previously characterized trans-Golgi network syntaxin proteins SYP41 and SYP51. An enhanced yellow fluorescent protein (EYFP)-RabA4b fusion protein specifically localizes to the tips of growing root hair cells in Arabidopsis thaliana. Tip-localized EYFP-RabA4b disappears in mature root hair cells that have stopped expanding, and polar localization of the EYFP-RabA4b is disrupted by latrunculin B treatment. Loss of tip localization of EYFP-RabA4b was correlated with inhibition of expansion; upon washout of the inhibitor, root hair expansion recovered only after tip localization of the EYFP-RabA4b compartments was reestablished. Furthermore, in mutants with defective root hair morphology, EYFP-RabA4b was improperly localized or was absent from the tips of root hair cells. We propose that RabA4b regulates membrane trafficking through a compartment involved in the polarized secretion of cell wall components in plant cells.     

Role of Phosphatidylinositol 4-Phosphate (PI4P) and Its Binding Protein GOLPH3 in Hepatitis C Virus Secretion

Hepatitis C virus (HCV) RNA replicates within the ribonucleoprotein complex, assembled on the endoplasmic reticulum (ER)-derived membranous structures closely juxtaposed to the lipid droplets that facilitate the post-replicative events of virion assembly and maturation. It is widely believed that the assembled virions piggy-back onto the very low density lipoprotein particles for secretion. Lipid phosphoinositides are important modulators of intracellular trafficking. Golgi-localized phosphatidylinositol 4-phosphate (PI4P) recruits proteins involved in Golgi trafficking to the Golgi membrane and promotes anterograde transport of secretory proteins. Here, we sought to investigate the role of Golgi-localized PI4P in the HCV secretion process. Depletion of the Golgi-specific PI4P pool by Golgi-targeted PI4P phosphatase hSac1 K2A led to significant reduction in HCV secretion without any effect on replication. We then examined the functional role of a newly identified PI4P binding protein GOLPH3 in the viral secretion process. GOLPH3 is shown to maintain a tensile force on the Golgi, required for vesicle budding via its interaction with an unconventional myosin, MYO18A. Silencing GOLPH3 led to a dramatic reduction in HCV virion secretion, as did the silencing of MYO18A. The reduction in virion secretion was accompanied by a concomitant accumulation of intracellular virions, suggesting a stall in virion egress. HCV-infected cells displayed a fragmented and dispersed Golgi pattern, implicating involvement in virion morphogenesis. These studies establish the role of PI4P and its interacting protein GOLPH3 in HCV secretion and strengthen the significance of the Golgi secretory pathway in this process.     

Sphingomyelin synthases regulate protein trafficking and secretion

Sphingomyelin synthases (SMS1 and 2) represent a class of enzymes that transfer a phosphocholine moiety from phosphatidylcholine onto ceramide thus producing sphingomyelin and diacylglycerol (DAG). SMS1 localizes at the Golgi while SMS2 localizes both at the Golgi and the plasma membrane. Previous studies from our laboratory showed that modulation of SMS1 and, to a lesser extent, of SMS2 affected the formation of DAG at the Golgi apparatus. As a consequence, down-regulation of SMS1 and SMS2 reduced the localization of the DAG-binding protein, protein kinase D (PKD), to the Golgi. Since PKD recruitment to the Golgi has been implicated in cellular secretion through the trans golgi network (TGN), the effect of down-regulation of SMSs on TGN-to-plasma membrane trafficking was studied. Down regulation of either SMS1 or SMS2 significantly retarded trafficking of the reporter protein vesicular stomatitis virus G protein tagged with GFP (VSVG-GFP) from the TGN to the cell surface. Inhibition of SMSs also induced tubular protrusions from the trans Golgi network reminiscent of inhibited TGN membrane fission. Since a recent study demonstrated the requirement of PKD activity for insulin secretion in beta cells, we tested the function of SMS in this model. Inhibition of SMS significantly reduced insulin secretion in rat INS-1 cells. Taken together these results provide the first direct evidence that both enzymes (SMS1 and 2) are capable of regulating TGN-mediated protein trafficking and secretion, functions that are compatible with PKD being a down-stream target for SMSs in the Golgi.     

Interaction between the triglyceride lipase ATGL and the Arf1 activator GBF1

The Arf1 exchange factor GBF1 (Golgi Brefeldin A resistance factor 1) and its effector COPI are required for delivery of ATGL (adipose triglyceride lipase) to lipid droplets (LDs). Using yeast two hybrid, co-immunoprecipitation in mammalian cells and direct protein binding approaches, we report here that GBF1 and ATGL interact directly and in cells, through multiple contact sites on each protein. The C-terminal region of ATGL interacts with N-terminal domains of GBF1, including the catalytic Sec7 domain, but not with full-length GBF1 or its entire N-terminus. The N-terminal lipase domain of ATGL (called the patatin domain) interacts with two C-terminal domains of GBF1, HDS (Homology downstream of Sec7) 1 and HDS2. These two domains of GBF1 localize to lipid droplets when expressed alone in cells, but not to the Golgi, unlike the full-length GBF1 protein, which localizes to both. We suggest that interaction of GBF1 with ATGL may be involved in the membrane trafficking pathway mediated by GBF1, Arf1 and COPI that contributes to the localization of ATGL to lipid droplets.     

PRAF1: a Golgi complex transmembrane protein that interacts with viruses.

Prenylated Rab acceptor domain family member 1 (PRAF1), a transmembrane protein whose precise function is unknown, localizes to the Golgi complex, post-Golgi vesicles, lipid rafts, endosomes, and the plasma membrane. VAMP2 and Rab3A are SNARE proteins that interact with PRAF1, and, as part of a SNARE complex, PRAF1 may function in the regulation of docking and fusion of transport vesicles both in the Golgi complex and at the plasma membrane. Alternately, PRAF1 may function as a sorting protein in the Golgi complex. In addition to interacting with SNARE proteins, PRAF1 interacts with rotaviral, retroviral, and herpes viral proteins. The function of viral protein interaction is unknown, but PRAF1 may enhance rotaviral and retroviral assembly. In contrast, PRAF1 may inhibit the herpes virus life cycle.     

Glucosylceramide Biosynthesis is Involved in Golgi Morphology and Protein Secretion in Plant Cells

Lipids have an established role as structural components of membranes or as signalling molecules, but their role as molecular actors in protein secretion is less clear. The complex sphingolipid glucosylceramide (GlcCer) is enriched in the plasma membrane and lipid microdomains of plant cells, but compared to animal and yeast cells, little is known about the role of GlcCer in plant physiology. We have investigated the influence of GlcCer biosynthesis by glucosylceramide synthase (GCS) on the efficiency of protein transport through the plant secretory pathway and on the maintenance of normal Golgi structure. We determined that GlcCer is synthesized at the beginning of the plant secretory pathway [mainly endoplasmic reticulum (ER)] and that d ,l ‐threo‐1‐phenyl‐2‐decanoyl amino‐3‐morpholino‐propanol (PDMP) is a potent inhibitor of plant GCS activity in vitro and in vivo. By an in vivo confocal microscopy approach in tobacco leaves infiltrated with PDMP, we showed that the decrease in GlcCer biosynthesis disturbed the transport of soluble and membrane secretory proteins to the cell surface, as these proteins were partly retained intracellularly in the ER and/or Golgi. Electron microscopic observations of Arabidopsis thaliana root cells after high‐pressure freezing and freeze substitution evidenced strong morphological changes in the Golgi bodies, pointing to a link between decreased protein secretion and perturbations of Golgi structure following inhibition of GlcCer biosynthesis in plant cells.     

RPA, a class II ARFGAP protein, activates ARF1 and U5 and plays a role in root hair development in Arabidopsis

The polar growth of plant cells depends on the secretion of a large amount of membrane and cell wall materials at the growing tip to sustain rapid growth. Small GTP-binding proteins, such as Rho-related GTPases from plants and ADP-ribosylation factors (ARFs), have been shown to play important roles in polar growth via regulating intracellular membrane trafficking. To investigate the role of membrane trafficking in plant development, a Dissociation insertion line that disrupted a putative ARF GTPase-activating protein (ARFGAP) gene, AT2G35210, was identified in Arabidopsis (Arabidopsis thaliana). Phenotypic analysis showed that the mutant seedlings developed isotropically expanded, short, and branched root hairs. Pollen germination in vitro indicated that the pollen tube growth rate was slightly affected in the mutant. AT2G35210 is specifically expressed in roots, pollen grains, and pollen tubes; therefore, it is designated as ROOT AND POLLEN ARFGAP (RPA). RPA encodes a protein with an N-terminal ARFGAP domain. Subcellular localization experiments showed that RPA is localized at the Golgi complexes via its 79 C-terminal amino acids. We further showed that RPA possesses ARF GTPase-activating activity and specifically activates Arabidopsis ARF1 and ARF1-like protein U5 in vitro. Furthermore, RPA complemented Saccharomyces cerevisiae glo3Delta gcs1Delta double mutant, which suggested that RPA functions as an ARFGAP during vesicle transport between the Golgi and the endoplasmic reticulum. Together, we demonstrated that RPA plays a role in root hair and pollen tube growth, most likely through the regulation of Arabidopsis ARF1 and ARF1-like protein U5 activity.     

The odyssey of hsp60 from tumor cells to other destinations includes plasma membrane-associated stages and Golgi and exosomal protein-trafficking modalities

Background

In a previous work we showed for the first time that human tumor cells secrete Hsp60 via exosomes, which are considered immunologically active microvesicles involved in tumor progression. This finding raised questions concerning the route followed by Hsp60 to reach the exosomes, its location in them, and whether Hsp60 can be secreted also via other mechanisms, e.g., by the Golgi. We addressed these issues in the work presented here.

Principal Findings

We found that Hsp60 localizes in the tumor cell plasma membrane, is associated with lipid rafts, and ends up in the exosomal membrane. We also found evidence that Hsp60 localizes in the Golgi apparatus and its secretion is prevented by an inhibitor of this organelle.

Conclusions/Significance

We propose a multistage process for the translocation of Hsp60 from the inside to the outside of the cell that includes a combination of protein traffic pathways and, ultimately, presence of the chaperonin in the circulating blood. The new information presented should help in designing future strategies for research and for developing diagnostic-monitoring means useful in clinical oncology.     

Cell growth-dependent coordination of lipid signaling and glycosylation is mediated by interactions between Sac1p and Dpm1p

The integral membrane lipid phosphatase Sac1p regulates local pools of phosphatidylinositol-4-phosphate (PtdIns(4)P) at endoplasmic reticulum (ER) and Golgi membranes. PtdIns(4)P is important for Golgi trafficking, yet the significance of PtdIns(4)P for ER function is unknown. It also remains unknown how localization of Sac1p to distinct organellar membranes is mediated. Here, we show that a COOH-terminal region in yeast Sac1p is crucial for ER targeting by directly interacting with dolicholphosphate mannose synthase Dpm1p. The interaction with Dpm1p persists during exponential cell division but is rapidly abolished when cell growth slows because of nutrient limitation, causing translocation of Sac1p to Golgi membranes. Cell growth-dependent shuttling of Sac1p between the ER and the Golgi is important for reciprocal control of PtdIns(4)P levels at these organelles. The fraction of Sac1p resident at the ER is also required for efficient dolichol oligosaccharide biosynthesis. Thus, the lipid phosphatase Sac1p may be a key regulator, coordinating the secretory capacity of ER and Golgi membranes in response to growth conditions.     

IRREGULAR TRICHOME BRANCH 2 (ITB2) encodes a putative aminophospholipid translocase that regulates trichome branch elongation in Arabidopsis

The P4 ATPase family in Arabidopsis consists of 12 members that encode putative aminophospholipid translocases (ALA1–12). Until recently, no mutations in these genes have been shown to cause a visible phenotype, although reduced expression of ALA1 in transgenic plants expressing an antisense construct has been shown to result in reduced plant size when plants were grown under cold conditions. During a genetic screen for mutations that affect trichome shape, we isolated several alleles of the irregular trichome branch 2 ( itb2 ) mutation. Subsequent positional cloning of this locus showed that ITB2 encoded ALA3 . Phenotypic and genetic analyses of multiple itb2 alleles, including the T-DNA insertion alleles, showed that the loss of ITB2 / ALA3 function leads to aberrant trichome expansion, reduced primary root growth and longer root hairs. We also found that itb2 / ala3 mutant pollen does not grow as well as wild-type pollen, leading to severe segregation distortion. Our results suggest that aminophospholipid translocases play an important role in the polar growth of plant cells, which is consistent with the proposed role of ALA3 in membrane trafficking. Furthermore, itb2 / ala3 mutants provide a convenient visible phenotype for further genetic analysis of the ALA family in Arabidopsis.     

Protein kinase D 3 is localized in vesicular structures and interacts with vesicle-associated membrane protein 2

Protein kinase D localizes in the Golgi and regulates protein transport from the Golgi to the plasma membrane. In the present study, we found that PKD3, a novel member of the PKD family, and its fluorescent protein fusions localized in the Golgi and in the vesicular structures that are in part marked by endosome markers. Fluorescent recovery after photobleaching (FRAP) showed that the PKD3-associated vesicular structures were constantly forming and dissolving, reflecting active subcellular structures. FRAP on plasma membrane-located PKD3 indicated a slower recovery of PKD3 fluorescent signal compared to those of PKC isoforms, implying a different targeting mechanism at the plasma membrane. VAMP2, the vesicle-localized v-SNARE, was later identified as a novel binding partner of PKD3 through yeast two-hybrid screening. PKD3 directly interacted with VAMP2 in vitro and in vivo, and colocalized in part with VAMP2 vesicles in cells. PKD3 did not phosphorylate VAMP-GFP and the purified GST-VAMP2 protein in in vitro phosphorylation assays. Rather, PKD3 was found to promote the recruitment of VAMP2 vesicles to the plasma membrane in response to PMA, while the kinase dead PKD3 abolished this effect. Thus, the kinase activity of PKD3 was required for PMA-induced plasma membrane trafficking of VAMP2. In summary, our findings suggest that PKD3 localizes to vesicular structures that are part of the endocytic compartment. The vesicular distribution may be attributed in part to the direct interaction between PKD3 and vesicle-associated membrane protein VAMP2, through which PKD3 may regulate VAMP2 vesicle trafficking by facilitating its recruitment to the target membrane.     

Phosphoinositide 3-kinase �� regulates membrane fission of Golgi carriers for selective cytokine secretion

Phosphoinositide 3-kinase (PI3K) p110 isoforms are membrane lipid kinases classically involved in signal transduction. Lipopolysaccharide (LPS)-activated macrophages constitutively and abundantly secrete proinflammatory cytokines including tumor necrosis factor-α (TNF). Loss of function of the p110δ isoform of PI3K using inhibitors, RNA-mediated knockdown, or genetic inactivation in mice abolishes TNF trafficking and secretion, trapping TNF in tubular carriers at the trans-Golgi network (TGN). Kinase-active p110δ localizes to the Golgi complex in LPS-activated macrophages, and TNF is loaded into p230-labeled tubules, which cannot undergo fission when p110δ is inactivated. Similar blocks in fission of these tubules and in TNF secretion result from inhibition of the guanosine triphosphatase dynamin 2. These findings demonstrate a new function for p110δ as part of the membrane fission machinery required at the TGN for the selective trafficking and secretion of cytokines in macrophages.     

Caenorhabditis elegans SNAP-29 is required for organellar integrity of the endomembrane system and general exocytosis in intestinal epithelial cells

It is generally accepted that soluble N-ethylmaleimide-sensitive factor attachment protein receptors mediate the docking and fusion of transport intermediates with target membranes. Our research identifies Caenorhabditis elegans homologue of synaptosomal-associated protein 29 (SNAP-29) as an essential regulator of membrane trafficking in polarized intestinal cells of living animals. We show that a depletion of SNAP-29 blocks yolk secretion and targeting of apical and basolateral plasma membrane proteins in the intestinal cells and results in a strong accumulation of small cargo-containing vesicles. The loss of SNAP-29 also blocks the transport of yolk receptor RME-2 to the plasma membrane in nonpolarized oocytes, indicating that its function is required in various cell types. SNAP-29 is essential for embryogenesis, animal growth, and viability. Functional fluorescent protein-tagged SNAP-29 mainly localizes to the plasma membrane and the late Golgi, although it also partially colocalizes with endosomal proteins. The loss of SNAP-29 leads to the vesiculation/fragmentation of the Golgi and endosomes, suggesting that SNAP-29 is involved in multiple transport pathways between the exocytic and endocytic organelles. These observations also suggest that organelles comprising the endomembrane system are highly dynamic structures based on the balance between membrane budding and fusion and that SNAP-29-mediated fusion is required to maintain proper organellar morphology and functions.     

Growth control of Golgi phosphoinositides by reciprocal localization of sac1 lipid phosphatase and pik1 4-kinase

Compartment-specific control of phosphoinositide lipids is essential for cell function. The Sac1 lipid phosphatase regulates endoplasmic reticulum (ER) and Golgi phosphatidylinositol-4-phosphate [PI(4)P] in response to nutrient levels and cell growth stages. During exponential growth, Sac1p interacts with Dpm1p at the ER but shuttles to the Golgi during starvation. Here, we report that a C-terminal region in Sac1p is required for retention in the perinuclear ER, whereas the N-terminal domain is responsible for Golgi localization. We also show that starvation-induced shuttling of Sac1p to the Golgi depends on the coat protein complex II and the Rer1 adaptor protein. Starvation-induced shuttling of Sac1p to the Golgi specifically eliminates a pool of PI(4)P generated by the lipid kinase Pik1p. In addition, absence of nutrients leads to a rapid dissociation of Pik1p, together with its non-catalytical subunit Frq1p, from Golgi membranes. Reciprocal rounds of association/dissociation of the Sac1p lipid phosphatase and the Pik1p/Frq1p lipid kinase complex are responsible for growth-dependent control of Golgi phosphoinositides. Sac1p and Pik1p/Frq1p are therefore elements of a unique machinery that synchronizes ER and Golgi function in response to different growth conditions.     

Imaging phosphatidylinositol 4-phosphate dynamics in living plant cells

Polyphosphoinositides represent a minor group of phospholipids, accounting for less than 1% of the total. Despite their low abundance, these molecules have been implicated in various signalling and membrane trafficking events. Phosphatidylinositol 4-phosphate (PtdIns4 P ) is the most abundant polyphosphoinositide. ³²Pi-labelling studies have shown that the turnover of PtdIns4 P is rapid, but little is known about where in the cell or plant this occurs. Here, we describe the use of a lipid biosensor that monitors PtdIns4 P dynamics in living plant cells. The biosensor consists of a fusion between a fluorescent protein and a lipid-binding domain that specifically binds PtdIns4 P , i.e. the pleckstrin homology domain of the human protein phosphatidylinositol-4-phosphate adaptor protein-1 (FAPP1). YFP–PH_FAPP1 was expressed in four plant systems: transiently in cowpea protoplasts, and stably in tobacco BY-2 cells, Medicago truncatula roots and Arabidopsis thaliana seedlings. All systems allowed YFP–PH_FAPP1 expression without detrimental effects. Two distinct fluorescence patterns were observed: labelling of motile punctate structures and the plasma membrane. Co-expression studies with organelle markers revealed strong co-labelling with the Golgi marker STtmd–CFP, but not with the endocytic/pre-vacuolar marker GFP–AtRABF2b. Co-expression with the Ptdins3 P biosensor YFP–2 × FYVE revealed totally different localization patterns. During cell division, YFP–PH_FAPP1 showed strong labelling of the cell plate, but PtdIns3 P was completely absent from the newly formed cell membrane. In root hairs of M. truncatula and A. thaliana , a clear PtdIns4 P gradient was apparent in the plasma membrane, with the highest concentration in the tip. This only occurred in growing root hairs, indicating a role for PtdIns4 P in tip growth.     

YSK1 is activated by the Golgi matrix protein GM130 and plays a role in cell migration through its substrate 14-3-3zeta

The Golgi apparatus has long been suggested to be important for directing secretion to specific sites on the plasma membrane in response to extracellular signaling events. However, the mechanisms by which signaling events are coordinated with Golgi apparatus function remain poorly understood. Here, we identify a scaffolding function for the Golgi matrix protein GM130 that sheds light on how such signaling events may be regulated. We show that the mammalian Ste20 kinases YSK1 and MST4 target to the Golgi apparatus via the Golgi matrix protein GM130. In addition, GM130 binding activates these kinases by promoting autophosphorylation of a conserved threonine within the T-loop. Interference with YSK1 function perturbs perinuclear Golgi organization, cell migration, and invasion into type I collagen. A biochemical screen identifies 14-3-3zeta as a specific substrate for YSK1 that localizes to the Golgi apparatus, and potentially links YSK1 signaling at the Golgi apparatus with protein transport events, cell adhesion, and polarity complexes important for cell migration.     

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.