The AtVAM3 Encodes a Syntaxin-related Molecule Implicated in the Vacuolar Assembly in Arabidopsis thaliana

Abstract

The vacuole constitutes a large compartment in plant and fungal cells. The VAM3 gene of Saccharomyces cerevisiae encodes a syntaxin-related protein required for vacuolar assembly. An Arabidopsis thaliana cDNA library, designed for expression in S. cerevisiae, was screened for cDNAs able to complement defective vacuolar assembly of the ⌬vam3 mutation. One cDNA, encoding a 33-kDa protein with structural similarities to the other syntaxins, was identified. The product of AtVAM3 (AtVam3p) was expressed in various tissues including roots, leaves, inflorescence stems, flower buds, and young siliques. The AtVAM3 transcripts were abundant in undifferentiated cells in the meristematic region. AtVam3p fractionated predominantly to an 8,000 ؋ g pellet fraction where a vacuolar membrane protein H ؉ -translocating inorganic pyrophosphatase (H ؉ -PPase) also fractionated. Immunoelectron microscopy showed that AtVam3p was localized to restricted regions on the vacuolar membranes. We propose that AtVam3p provides the t-SNARE function in the vacuolar assembly in A. thaliana.The vacuole in plant and fungal cells constitutes a large compartment with various physiological functions (1, 2). The vacuole serves as a storage compartment for various primary and secondary metabolites including amino acids, organic acids, and sugars. It is a lytic compartment as well, sharing common features with lysosomes of mammalian cells. It also functions in homeostatic regulation of cytosolic ions by transporting small molecules across the vacuolar membrane through various transporters. One of the most prominent features of the plant vacuole is its large volume. In fully matured plant cells, it occupies over 90% of cell volume. Individual plants need to expand their stems, leaves, and roots to acquire resources from the environment including light energy, minerals, and water. This expansion in plant body size, however, would be costly if it were accomplished by cell division or by synthesis of cytosolic material. In fact, cell enlargement is mostly attributable to the increase in vacuolar volume. Therefore, the plant vacuole provides a space-filling compartment (3).While the physiological importance of the plant vacuole is clear and many of the molecules that make up this compartment have been well characterized, the molecular mechanisms involved in its assembly are largely unknown. In contrast, vacuolar biogenesis and assembly in the unicellular organism Saccharomyces cerevisiae are fairly well understood. Our previous genetic studies showed that nine VAM genes (for vacuolar morphology) are involved in the assembly of yeast vacuolar compartments (4). We have also shown that the yeast VAM3 gene encodes a member of the syntaxin family (5), the key molecules regulating vesicular transport (6, 7). Syntaxins are membrane receptors on the target membrane (t-SNARE) that interact with the other molecules on the transport vesicles (v-SNARE). Specific interactions between the t-SNARE and v-SNARE molecules ensure fusion of the transport vesicles with their ta...

Identification and Dynamics of Arabidopsis Adaptor Protein-2 Complex and Its Involvement in Floral Organ Development

“…Colocalization of AP2M-GFP fluorescence with FM4-64 was also observed in the cotyledon epidermal cells of AP2M-GFP ap2m plants (see Supplemental Figure 1 online). Subcellular fractionation of AP2M-GFP ap2m seedlings revealed that a major part of the AP2M-GFP protein was found in the soluble fraction (S100), which accumulated the vacuolar Cys protease aleurain (ALEU) (Rogers et al, 1997;Watanabe et al, 2004), whereas a minor part of the AP2M-GFP protein was detected in the microsome fraction (P100), which accumulated the vacuolar SNARE protein VAM3 (Sato et al, 1997) (Figure 1B). These results suggest that the AP2M protein is predominantly accumulated in the cytosol and is also localized to the plasma membrane.…”

“…The pellet (P100, microsome fraction) was resuspended in 4 mL of chopping buffer, and the supernatant was used as the S100 (soluble) fraction. Aliquots of the total, P10, P100, and S100 suspensions were separated by SDS-PAGE and used for immunoblot analysis using anti-VAM3 antibody (Sato et al, 1997) and anti-ALEU antibody (Rogers et al, 1997). The remaining suspensions were mixed with magnetic beads conjugated with anti-GFP antibody (Miltenyi Biotec), incubated on ice for 30 min, and then applied to m-Columns (Miltenyi Biotec) in a magnetic field.…”

Plant Cells Are Not Just Green Yeast

“…The AtVAM3 gene was isolated because of its ability to complement several phenotypes of the yeast vam3 mutant (Sato et al, 1997). Yeast Vam3p is a t-SNARE present in the vacuolar membrane, where it functions in the fusion of transport vesicles from several trafficking pathways with the vacuole.…”

Tansley Review No. 108

“…However, the pattern of At-Vam3 accumulation, prevalent on mature vacuolar membranes (Sato et al, 1997), is clearly different from that of At-Pep12. Conceicao et al (1997) reported that At-Pep12 associated with a prevacuolar compartment thought to be an intermediary step in vesicle transport from the transGolgi network (TGN).…”

“…Several of these mutants have now been used in complementation screens with plant cDNA libraries. The Arabidopsis counterparts to the yeast t-SNAREs Pep12p and Vam3p (Bassham et al, 1995 ;BarPeled & Raikhel, 1997 ;Sato et al, 1997), and at least one group of Arabidopsis and Nicotiana GTPase proteins (Bednarek et al, 1994 ;Takeuchi et al, 1998) were first characterized in this manner.…”