The Plant Vesicle-associated SNARE AtVTI1a Likely Mediates Vesicle Transport from the Trans-Golgi Network to the Prevacuolar Compartment

Membrane traffic in eukaryotic cells relies on recognition between v-SNAREs on transport vesicles and t-SNAREs on target membranes. Here we report the identification of AtVTI1a and AtVTI1b, two Arabidopsis homologues of the yeast v-SNARE Vti1p, which is required for multiple transport steps in yeast. AtVTI1a and AtVTI1b share 60% amino acid identity with one another and are 32 and 30% identical to the yeast protein, respectively. By suppressing defects found in specific strains of yeast vti1 temperature-sensitive mutants, we show that AtVTI1a can substitute for Vti1p in Golgi-to-prevacuolar compartment (PVC) transport, whereas AtVTI1b substitutes in two alternative pathways: the vacuolar import of alkaline phosphatase and the so-called cytosol-to-vacuole pathway used by aminopeptidase I. Both AtVTI1a and AtVTI1b are expressed in all major organs of Arabidopsis. Using subcellular fractionation and immunoelectron microscopy, we show that AtVTI1a colocalizes with the putative vacuolar cargo receptor AtELP on the trans-Golgi network and the PVC. AtVTI1a also colocalizes with the t-SNARE AtPEP12p to the PVC. In addition, AtVTI1a and AtPEP12p can be coimmunoprecipitated from plant cell extracts. We propose that AtVTI1a functions as a v-SNARE responsible for targeting AtELP-containing vesicles from the trans-Golgi network to the PVC, and that AtVTI1b is involved in a different membrane transport process.     

The t-SNARE AtVAM3p resides on the prevacuolar compartment in Arabidopsis root cells

Protein cargo is trafficked between the organelles of the endomembrane system inside transport vesicles, a process mediated by integral membrane proteins called SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) that reside on the surface of the vesicle (v-SNAREs) and target membrane (t-SNAREs). In examining transport of cargo between the trans-Golgi network and the vacuole in Arabidopsis, we have previously characterized AtPEP12p as a t-SNARE residing on the prevacuolar compartment and AtVTI1a as a v-SNARE that interacts with AtPEP12p. Recently, we have begun to characterize AtVAM3p, another Arabidopsis t-SNARE that shows high sequence homology to AtPEP12p. We have found that AtVTI1a also interacts with AtVAM3p, suggesting a role for this t-SNARE in post-Golgi trafficking. AtVAM3p has been suggested to localize to the vacuolar membrane in Arabidopsis cells; however, using specific antisera and expression of epitope-tagged versions of each t-SNARE, we have discovered that AtVAM3p is found on the same prevacuolar structure as AtPEP12p in Arabidopsis root cells.     

AtVPS45 Is a Positive Regulator of the SYP41/SYP61/VTI12 SNARE Complex Involved in Trafficking of Vacuolar Cargo

We report a functional characterization of AtVPS45 (for vacuolar protein sorting 45), a protein from the Sec1/Munc18 family in Arabidopsis (Arabidopsis thaliana) that interacts at the trans-Golgi network (TGN) with the SYP41/SYP61/VTI12 SNARE complex. A null allele of AtVPS45 was male gametophytic lethal, whereas stable RNA interference lines with reduced AtVPS45 protein levels had stunted growth but were viable and fertile. In the silenced lines, we observed defects in vacuole formation that correlated with a reduction in cell expansion and with autophagy-related defects in nutrient turnover. Moreover, transport of vacuolar cargo with carboxy-terminal vacuolar sorting determinants was blocked in the silenced lines, suggesting that AtVPS45 functions in vesicle trafficking to the vacuole. These trafficking defects are similar to those observed in vti12 mutants, supporting a functional relationship between AtVPS45 and VTI12. Consistent with this, we found a decrease in SYP41 protein levels coupled to the silencing of AtVPS45, pointing to instability and malfunction of the SYP41/SYP61/VTI12 SNARE complex in the absence of its cognate Sec1/Munc18 regulator. Based on its localization on the TGN, we hypothesized that AtVPS45 could be involved in membrane fusion of retrograde vesicles recycling vacuolar trafficking machinery. Indeed, in the AtVPS45-silenced plants, we found a striking alteration in the subcellular fractionation pattern of vacuolar sorting receptors, which are required for sorting of carboxy-terminal vacuolar sorting determinant-containing cargo. We propose that AtVPS45 is essential for recycling of the vacuolar sorting receptors back to the TGN and that blocking this step underlies the defects in vacuolar cargo trafficking observed in the silenced lines.     

An Arabidopsis VPS45p Homolog Implicated in Protein Transport to the Vacuole

The Sec1p family of proteins is required for vesicle-mediated protein trafficking between various organelles of the endomembrane system. This family includes Vps45p, which is required for transport to the vacuole in yeast (Saccharomyces cerevisiae). We have isolated a cDNA encoding a VPS45 homolog from Arabidopsis thaliana (AtVPS45). The cDNA is able to complement both the temperature-sensitive growth defect and the vacuolar-targeting defect of a yeast vps45 mutant, indicating that the two proteins are functionally related. AtVPS45p is a peripheral membrane protein that associates with microsomal membranes. Sucrose-density gradient fractionation demonstrated that AtVPS45p co-fractionates with AtELP, a potential vacuolar protein sorting receptor, implying that they may reside on the same membrane populations. These results indicate that AtVPS45p is likely to function in the transport of proteins to the vacuole in plants.     

Two syntaxin homologues in the TGN/endosomal system of yeast.

Intracellular membrane traffic is thought to be regulated in part by SNAREs, integral membrane proteins on transport vesicles (v-SNAREs) and target organelles (t-SNAREs) that bind to each other and mediate bilayer fusion. All known SNARE-mediated fusion events involve a member of the syntaxin family of t-SNAREs. Sequence comparisons identify eight such proteins encoded in the yeast genome, of which six have been characterized. We describe here the remaining two, Tlg1p and Tlg2p. These have the expected biochemical properties of t-SNAREs, and are located in separable compartments which correspond to a putative early endosome and the yeast equivalent of the TGN, respectively. They co-precipitate with the v-SNARE Vti1p, which is implicated in Golgi-endosome traffic and, remarkably, binds to five different syntaxins. Tlg1p also binds the plasma membrane v-SNARE Snc1p. Both Tlg1p and Tlg2p are required for efficient endocytosis and to maintain normal levels of TGN proteins. However, neither is required for intra-Golgi traffic. Since no further syntaxins have been identified in yeast, this implies that the Golgi apparatus can function with a single syntaxin, Sed5p.     

The Saccharomyces cerevisiae v-SNARE Vti1p Is Required for Multiple Membrane Transport Pathways to the Vacuole

The interaction between v-SNAREs on transport vesicles and t-SNAREs on target membranes is required for membrane traffic in eukaryotic cells. Here we identify Vti1p as the first v-SNARE protein found to be required for biosynthetic traffic into the yeast vacuole, the equivalent of the mammalian lysosome. Certain vti1-ts yeast mutants are defective in alkaline phosphatase transport from the Golgi to the vacuole and in targeting of aminopeptidase I from the cytosol to the vacuole. VTI1 interacts genetically with the vacuolar t-SNARE VAM3, which is required for transport of both alkaline phosphatase and aminopeptidase I to the vacuole. The v-SNARE Nyv1p forms a SNARE complex with Vam3p in homotypic vacuolar fusion; however, we find that Nyv1p is not required for any of the three biosynthetic pathways to the vacuole. v-SNAREs were thought to ensure specificity in membrane traffic. However, Vti1p also functions in two additional membrane traffic pathways: Vti1p interacts with the t-SNAREs Pep12p in traffic from the TGN to the prevacuolar compartment and with Sed5p in retrograde traffic to the cis-Golgi. The ability of Vti1p to mediate multiple fusion steps requires additional proteins to ensure specificity in membrane traffic.     

Vps45p stabilizes the syntaxin homologue Tlg2p and positively regulates SNARE complex formation.

Sec1p-like/Munc-18 (SM) proteins bind to t-SNAREs and inhibit ternary complex formation. Paradoxically, the absence of SM proteins does not result in constitutive membrane fusion. Here, we show that in yeast cells lacking the SM protein Vps45p, the t-SNARE Tlg2p is down-regulated, to undetectable levels, by rapid proteasomal degradation. In the absence of Vps45p, Tlg2p can be stabilized through abolition of proteasome activity. Surprisingly, the stabilized Tlg2p was targeted to the correct intracellular location. However, the stabilized Tlg2p is non-functional and unable to bind its cognate SNARE binding partners, Tlg1p and Vti1p, in the absence of Vps45p. A truncation mutant lacking the first 230 residues of Tlg2p no longer bound Vps45p but was able to form complexes with Tlg1p and Vti1p in the absence of the SM protein. These data provide us with two valuable insights into the function of SM proteins. First, SM proteins act as chaperone-like molecules for their cognate t-SNAREs. Secondly, SM proteins play an essential role in the activation process allowing their cognate t-SNARE to participate in ternary complex formation.     

A t-SNARE of the endocytic pathway must be activated for fusion.

The t-SNARE in a late Golgi compartment (Tlg2p) syntaxin is required for endocytosis and localization of cycling proteins to the late Golgi compartment in yeast. We show here that Tlg2p assembles with two light chains, Tlg1p and Vti1p, to form a functional t-SNARE that mediates fusion, specifically with the v-SNAREs Snc1p and Snc2p. In vitro, this t-SNARE is inert, locked in a nonfunctional state, unless it is activated for fusion. Activation can be mediated by a peptide derived from the v-SNARE, which likely bypasses additional regulatory proteins in the cell. Locking t-SNAREs creates the potential for spatial and temporal regulation of fusion by signaling processes that unleash their fusion capacity.     

A novel SNARE complex implicated in vesicle fusion with the endoplasmic reticulum.

Intracellular vesicular traffic is controlled in part by v- and t-SNAREs, integral membrane proteins which allow specific interaction and fusion between vesicles (v-SNAREs) and their target membranes (t-SNAREs). In yeast, retrograde transport from the Golgi complex to the ER is mediated by the ER t-SNARE Ufe1p, and also requires two other ER proteins, Sec20p and Tip20p, which bind each other. Although Sec20p is not a typical SNARE, we show that both it and Tip20p can be co-precipitated with Ufe1p, and that a growth-inhibiting mutation in Ufe1p can be compensated by a mutation in Sec20p. Furthermore, Sec22p, a v-SNARE implicated in forward transport from ER to Golgi, co-precipitates with Ufe1p and Sec20p, and SEC22 acts as an allele-specific multicopy suppressor of a temperature-sensitive ufe1 mutation. These results define a new functional SNARE complex, with features distinct from the plasma membrane and cis-Golgi complexes previously identified. They also show that a single v-SNARE can be involved in both anterograde and retrograde transport, which suggests that the mere presence of a particular v-SNARE may not be sufficient to determine the preferred target for a transport vesicle.     

A Vacuolar v–t-SNARE Complex, the Predominant Form In Vivo and on Isolated Vacuoles, Is Disassembled and Activated for Docking and Fusion

Homotypic vacuole fusion in yeast requires Sec18p (N-ethylmaleimide–sensitive fusion protein [NSF]), Sec17p (soluble NSF attachment protein [α-SNAP]), and typical vesicle (v) and target membrane (t) SNAP receptors (SNAREs). We now report that vacuolar v- and t-SNAREs are mainly found with Sec17p as v–t-SNARE complexes in vivo and on purified vacuoles rather than only transiently forming such complexes during docking, and disrupting them upon fusion. In the priming reaction, Sec18p and ATP dissociate this v–t-SNARE complex, accompanied by the release of Sec17p. SNARE complex structure governs each functional aspect of priming, as the v-SNARE regulates the rate of Sec17p release and, in turn, Sec17p-dependent SNARE complex disassembly is required for independent function of the two SNAREs. Sec17p physically and functionally interacts largely with the t-SNARE. (a) Antibodies to the t-SNARE, but not the v-SNARE, block Sec17p release. (b) Sec17p is associated with the t-SNARE in the absence of v-SNARE, but is not bound to the v-SNARE without t-SNARE. (c) Vacuoles with t-SNARE but no v-SNARE still require Sec17p/Sec18p priming, whereas their fusion partners with v-SNARE but no t-SNARE do not. Sec18p thus acts, upon ATP hydrolysis, to disassemble the v–t-SNARE complex, prime the t-SNARE, and release the Sec17p to allow SNARE participation in docking and fusion. These studies suggest that the analogous ATP-dependent disassembly of the 20-S complex of NSF, α-SNAP, and v- and t-SNAREs, which has been studied in detergent extracts, corresponds to the priming of SNAREs for docking rather than to the fusion of docked membranes.     

Ric1p and the Ypt6p GTPase function in a common pathway required for localization of trans-Golgi network membrane proteins

In Saccharomyces cerevisiae, clathrin is necessary for localization of trans-Golgi network (TGN) membrane proteins, a process that involves cycling of TGN proteins between the TGN and endosomes. To characterize further TGN protein localization, we applied a screen for mutations that cause severe growth defects in combination with a temperature-sensitive clathrin heavy chain. This screen yielded a mutant allele of RIC1. Cells carrying a deletion of RIC1 (ric1Delta) mislocalize TGN membrane proteins Kex2p and Vps10p to the vacuole. Delivery to the vacuole occurs in ric1Delta cells also harboring end3Delta to block endocytosis, indicative of a defect in retrieval to the TGN rather than sorting to endosomes. SYS1, originally discovered as a multicopy suppressor of defects caused by the absence of the Rab GTPase YPT6, was identified as a multicopy suppressor of ric1Delta. Further comparison of ric1Delta and ypt6Delta cells demonstrated identical phenotypes. Multicopy plasmids expressing v-SNAREs Gos1p or Ykt6p, but not other v- and t-SNAREs, partially suppressed phenotypes of ric1Delta and ypt6Delta cells. SLY1-20, a dominant activator of the cis-Golgi network t-SNARE Sed5p, also functioned as a multicopy suppressor. Because Gos1p and Ykt6p interact with Sed5p, these results raise the possibility that TGN membrane protein localization requires Ric1p- and Ypt6p-dependent retrieval to the cis-Golgi network.     

The AtC-VPS protein complex is localized to the tonoplast and the prevacuolar compartment in arabidopsis

Plant cells contain several types of vacuoles with specialized functions. Although the biogenesis of these organelles is well understood at the morphological level, the machinery involved in plant vacuole formation is largely unknown. We have recently identified an Arabidopsis mutant, vcl1, that is deficient in vacuolar formation. VCL1 is homologous to a protein that regulates membrane fusion at the tonoplast in yeast. On the basis of these observations, VCL1 is predicted to play a direct role in vacuolar biogenesis and vesicular trafficking to the vacuole in plants. In this work, we show that VCL1 forms a complex with AtVPS11 and AtVPS33 in vivo. These two proteins are homologues of proteins that have a well-characterized role in membrane fusion at the tonoplast in yeast. VCL1, AtVPS11, and AtVPS33 are membrane-associated and cofractionate with tonoplast and denser endomembrane markers in subcellular fractionation experiments. Consistent with this, VCL1, AtVPS11, and AtVPS33 are found on the tonoplast and the prevacuolar compartment (PVC) by immunoelectron microscopy. We also show that a VCL1-containing complex includes SYP2-type syntaxins and is most likely involved in membrane fusion on both the PVC and tonoplast in vivo. VCL1, AtVPS11, and AtVPS33 are the first components of the vacuolar biogenesis machinery to be identified in plants.     

Ca(2+)-synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion

In nerve terminals, exocytosis is mediated by SNARE proteins and regulated by Ca(2+) and synaptotagmin-1 (syt). Ca(2+) promotes the interaction of syt with anionic phospholipids and the target membrane SNAREs (t-SNAREs) SNAP-25 and syntaxin. Here, we have used a defined reconstituted fusion assay to determine directly whether syt-t-SNARE interactions couple Ca(2+) to membrane fusion by comparing the effects of Ca(2+)-syt on neuronal (SNAP-25, syntaxin and synaptobrevin) and yeast (Sso1p, Sec9c and Snc2p) SNAREs. Ca(2+)-syt aggregated neuronal and yeast SNARE liposomes to similar extents via interactions with anionic phospholipids. However, Ca(2+)-syt was able to bind and stimulate fusion mediated by only neuronal SNAREs and had no effect on yeast SNAREs. Thus, Ca(2+)-syt regulates fusion through direct interactions with t-SNAREs and not solely through aggregation of vesicles. Ca(2+)-syt drove assembly of SNAP-25 onto membrane-embedded syntaxin, providing direct evidence that Ca(2+)-syt alters t-SNARE structure.     

Regulation of intracellular membrane trafficking and cell dynamics by syntaxin-6

Intracellular membrane trafficking along endocytic and secretory transport pathways plays a critical role in diverse cellular functions including both developmental and pathological processes. Briefly, proteins and lipids destined for transport to distinct locations are collectively assembled into vesicles and delivered to their target site by vesicular fusion. SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins are required for these events, during which v-SNAREs (vesicle SNAREs) interact with t-SNAREs (target SNAREs) to allow transfer of cargo from donor vesicle to target membrane. Recently, the t-SNARE family member, syntaxin-6, has been shown to play an important role in the transport of proteins that are key to diverse cellular dynamic processes. In this paper, we briefly discuss the specific role of SNAREs in various mammalian cell types and comprehensively review the various roles of the Golgi- and endosome-localized t-SNARE, syntaxin-6, in membrane trafficking during physiological as well as pathological conditions.     

TNO1 is involved in salt tolerance and vacuolar trafficking in Arabidopsis

The Arabidopsis (Arabidopsis thaliana) soluble N-ethylmaleimide-sensitive factor attachment protein receptor SYP41 is involved in vesicle fusion at the trans-Golgi network (TGN) and interacts with AtVPS45, SYP61, and VTI12. These proteins are involved in diverse cellular processes, including vacuole biogenesis and stress tolerance. A previously uncharacterized protein, named TNO1 (for TGN-localized SYP41-interacting protein), was identified by coimmunoprecipitation as a SYP41-interacting protein. TNO1 was found to localize to the TGN by immunofluorescence microscopy. A tno1 mutant showed increased sensitivity to high concentrations of NaCl, KCl, and LiCl and also to mannitol-induced osmotic stress. Localization of SYP61, which is involved in the salt stress response, was disrupted in the tno1 mutant. Vacuolar proteins were partially secreted to the apoplast in the tno1 mutant, suggesting that TNO1 is required for efficient protein trafficking to the vacuole. The tno1 mutant had delayed formation of the brefeldin A (BFA) compartment in cotyledons upon application of BFA, suggesting less efficient membrane fusion processes in the mutant. Unlike most TGN proteins, TNO1 does not relocate to the BFA compartment upon BFA treatment. These data demonstrate that TNO1 is involved in vacuolar trafficking and salt tolerance, potentially via roles in vesicle fusion and in maintaining TGN structure or identity.     

The Yeast v-SNARE Vti1p Mediates Two Vesicle Transport Pathways through Interactions with the t-SNAREs Sed5p and Pep12p

Membrane traffic in eukaryotic cells requires that specific v-SNAREs on transport vesicles interact with specific t-SNAREs on target membranes. We identified a novel Saccharomyces cerevisiae v-SNARE (Vti1p) encoded by the essential gene, VTI1. Vti1p interacts with the prevacuolar t-SNARE Pep12p to direct Golgi to prevacuolar traffic. vti1-1 mutant cells missorted and secreted the soluble vacuolar hydrolase carboxypeptidase Y (CPY) rapidly and reversibly when vti1-1 cells were shifted to the restrictive temperature. However, overexpression of Pep12p suppressed the CPY secretion defect exhibited by vti1-1 cells at 36°C. Characterization of a second vti1 mutant, vti1-11, revealed that Vti1p also plays a role in membrane traffic at a cis-Golgi stage. vti1-11 mutant cells displayed a growth defect and accumulated the ER and early Golgi forms of both CPY and the secreted protein invertase at the nonpermissive temperature. Overexpression of the yeast cis-Golgi t-SNARE Sed5p suppressed the accumulation of the ER form of CPY but did not lead to CPY transport to the vacuole in vti1-11 cells. Overexpression of Sed5p allowed growth in the absence of Vti1p. In vitro binding and coimmunoprecipitation studies revealed that Vti1p interacts directly with the two t-SNAREs, Sed5p and Pep12p. These data suggest that Vti1p plays a role in cis-Golgi membrane traffic, which is essential for yeast viability, and a nonessential role in the fusion of Golgi-derived vesicles with the prevacuolar compartment. Therefore, a single v-SNARE can interact functionally with two different t-SNAREs in directing membrane traffic in yeast.     

Promiscuity in Rab-SNARE interactions

Fusion of post-Golgi secretory vesicles with the plasma membrane in yeast requires the function of a Rab protein, Sec4p, and a set of v- and t-SNAREs, the Snc, Sso, and Sec9 proteins. We have tested the hypothesis that a selective interaction between Sec4p and the exocytic SNAREs is responsible for ensuring that secretory vesicles fuse with the plasma membrane but not with intracellular organelles. Assembly of Sncp and Ssop into a SNARE complex is defective in a sec4-8 mutant strain. However, Snc2p binds in vivo to many other syntaxin-like t-SNAREs, and binding of Sncp to the endosomal/Golgi t-SNARE Tlg2p is also reduced in sec4-8 cells. In addition, binding of Sncp to Ssop is reduced by mutations in two other Rab genes and four non-Rab genes that block the secretory pathway before the formation of secretory vesicles. In an alternate approach to look for selective Rab-SNARE interactions, we report that the nucleotide-free form of Sec4p coimmunoprecipitates with Ssop. However, Rab-SNARE binding is nonselective, because the nucleotide-free forms of six Rab proteins bind with similar low efficiency to three SNARE proteins, Ssop, Pep12p, and Sncp. We conclude that Rabs and SNAREs do not cooperate to specify the target membrane.     

Cytoplasm to vacuole trafficking of aminopeptidase I requires a t-SNARE-Sec1p complex composed of Tlg2p and Vps45p.

Aminopeptidase I (API) is imported into the yeast vacuole/lysosome by a constitutive non-classical vesicular transport mechanism, the cytoplasm to vacuole targeting (Cvt) pathway. Newly synthesized precursor API is sequestered in double-membrane cytoplasmic Cvt vesicles. The Cvt vesicles fuse with the vacuole, releasing single-membrane Cvt bodies containing proAPI into the vacuolar lumen, and maturation of API occurs when the Cvt body is degraded, releasing mature API. Under starvation conditions, API is transported to the vacuole by macroautophagy, an inducible, non-selective mechanism that shares many similarities with the Cvt pathway. Here we show that Tlg2p, a member of the syntaxin family of t-SNARE proteins, and Vps45p, a Sec1p homologue, are required in the constitutive Cvt pathway, but not in inducible macroautophagy. Fractionation and protease protection experiments indicate that Tlg2p is required prior to or at the step of API segregation into the Cvt vesicle. Thus, the early Vps45-Tlg2p-dependent step of the Cvt pathway appears to be mechanistically distinct from the comparable stage in macroautophagy. Vps45p associates with both the Tlg2p and Pep12p t-SNAREs, but API maturation is not blocked in a pep12(ts) mutant, indicating that Vps45p independently regulates the function of multiple t-SNARES at distinct trafficking steps.     

Cell-free transport from the trans-golgi network to late endosome requires factors involved in formation and consumption of clathrin-coated vesicles

Transport between the trans-Golgi network (TGN) and late endosome represents a conserved, clathrin-dependent sorting event that separates lysosomal from secretory cargo molecules and is also required for localization of integral membrane proteins to the TGN. Previously, we reported a cell-free reaction that reconstitutes transport from the yeast TGN to the late endosome/prevacuolar compartment (PVC) and requires the PVC t-SNARE Pep12p. Here, we report that factors required both for formation of clathrin-coated vesicles at the TGN (the Chc1p clathrin heavy chain and the Vps1p dynamin homolog) and for vesicle fusion at the PVC (the Vps21p rab protein and Vps45p SM (Sec1/Munc18) protein) are required for cell-free transport. The marker for TGN-PVC transport, Kex2p, is initially present in a clathrin-containing membrane compartment that is competent for delivery of Kex2p to the PVC. A Kex2p chimera containing the cytosolic tail (C-tail) of the vacuolar protein sorting receptor, Vps10p, is also efficiently transported to the PVC. Antibodies against the Kex2p and Vps10p C-tails selectively block transport of Kex2p and the Kex2-Vps10p chimera. The requirements for factors involved in vesicle formation and fusion, the identification of the donor compartment as a clathrin-containing membrane, and the need for accessibility of C-tail sequences argue that the TGN-PVC transport reaction involves selective incorporation of TGN cargo molecules into clathrin-coated vesicle intermediates. Further biochemical dissection of this reaction should help elucidate the molecular requirements and hierarchy of events in TGN-to-PVC sorting and transport.     

Three SAC1-like genes show overlapping patterns of expression in Arabidopsis but are remarkably silent during embryo development

In Saccharomyces cerevisiae, the SAC1 gene encodes a polyphosphoinositide phosphatase (PPIPase) that modulates the levels of phosphoinositides, which are key regulators of a number of signal transduction processes. SAC1p has been implicated in multiple cellular functions: actin cytoskeleton organization, secretory functions, inositol metabolism, ATP transport, and multiple-drug sensitivity. Here, we describe the characterization of three genes in Arabidopsis thaliana, AtSAC1a, AtSAC1b, and AtSAC1c, encoding proteins similar to those of yeast SAC1p. We demonstrated that the three AtSAC1 proteins are functional homologs of the yeast SAC1p because they can rescue the cold-sensitive and inositol auxotroph yeast sac1-null mutant strain. The fact that Arabidopsis and yeast SAC1 genes derived from a common ancestor suggests that this plant multigenic family is involved in the phosphoinositide pathway and in a range of cellular functions similar to those in yeast. Using GFP fusion experiments, we demonstrate that the three AtSAC1 proteins are targeted to the endoplasmic reticulum. Their expression patterns are overlapping, with at least two members expressed in each organ. Remarkably, AtSAC1 genes are not expressed during seed development, and therefore additional phosphatases are required to control phosphoinositide levels in seeds.