Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Apr;149(4):1668-78.
doi: 10.1104/pp.108.134361. Epub 2009 Feb 27.

AtVPS45 is a positive regulator of the SYP41/SYP61/VTI12 SNARE complex involved in trafficking of vacuolar cargo

Jan Zouhar  1 , Enrique Rojo, Diane C Bassham
Affiliations

AtVPS45 is a positive regulator of the SYP41/SYP61/VTI12 SNARE complex involved in trafficking of vacuolar cargo

Jan Zouhar et al. Plant Physiol. 2009 Apr.

Abstract

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.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
AtVPS45 is required for cell expansion. A, Structure of the AtVPS45 gene, with boxes representing coding regions. The triangles indicate the sites of the T-DNA insertions in the Atvps45 knockout mutants. B, Individuals from the T3 generation of four independent homozygous RNAi transgenic lines are shown, with wild-type plants for comparison. Line 1 corresponds to siVPS45-1a, line 3 to siVPS45-8b, and line 4 to siVPS45-10d. Below are shown immunoblots of total protein extracts from each line probed with antibodies against AtVPS45. The size estimated for the AtVPS45 band was 66 kD. C, Cells of siVPS45-10d plants are smaller than those of wild-type plants. Wild-type or siVPS45-10d leaves were stained with chlorazol black E and observed by microscopy. The top panels show epidermal cells, and the bottom panels show mesophyll cells. Bar = 50 μm. D, Wild-type or siVPS45-10d seeds were germinated in the dark for 4 d. Longitudinal sections of hypocotyls of the etiolated seedlings were stained with toluidine blue and observed by light microscopy. Bar = 100 μm. WT or Wt, Wild type; si10d, siVPS45-10d.
Figure 2.
Figure 2.
The AtVPS45-silenced lines have vacuolar defects. A, Cross sections of hypocotyls from 4-d-old etiolated wild-type or siVPS45-10d seedlings were analyzed by electron microscopy. B, The protein storage vacuoles of epidermal cells from embryos of wild-type, siVPS45-10d, and siVPS45-8b seeds were imaged by confocal microscopy. Bar = 5 μm. C, The first and second true leaves from 24-d-old plants grown on soil were excised, placed on water in 24-well plates, incubated in the dark, and imaged at the indicated days after detaching. Wt, Wild type; si10d, siVPS45-10d; si8b, siVPS45-8b.
Figure 3.
Figure 3.
The AtVPS45-silenced plants secrete the VAC2 cargo. A, Plants from the F1 generation of crosses between a line homozygous for VAC2 (L1) and siVPS45-1a or siVPS45-10d display terminated flower and shoot meristems. B, Plants from the F2 generation of crosses between siVPS45-1a or siVPS45-10d and the L1 line. The plants shown were homozygous for VAC2 and either wild type (Wt) or AtVPS45 silenced (si1a and si10d). Arrows indicate flowers without carpels, and arrowheads indicate terminated shoot meristems.
Figure 4.
Figure 4.
The AtVPS45-silenced plants secrete ctVSD-containing cargo. A, Lines expressing GFP-CHI and ALEU-GFP were crossed with wild-type and siVPS45-10d plants, and the adaxial side of leaves from F1 plants was analyzed by confocal microscopy. Bars = 10 μm. B, Samples of apoplastic fluids (Apo) collected from rosette leaves of wild-type and siVPS45-10d plants were analyzed with the indicated antibodies. Total proteins (Total) from leaves of the same plants were analyzed to determine the expression levels of the proteins. The precursor (p), intermediate (i), and mature (m) forms of CPY are marked. C, The apoplastic fluids and total proteins from rosette leaves of the indicated mutants were analyzed with anti-AtAleurain antibodies (ALEU). evti11, enhanced vti11; evti12, enhanced vti12 (Sanmartin et al., 2007). Positions of molecular mass markers (in kD) are shown at left in B and C. Wt, Wild type; si10d, siVPS45-10d.
Figure 5.
Figure 5.
AtVPS45 is required for the stability of SYP41 and the proper subcellular distribution of VSRs. A, Protein samples from wild-type and AtVPS45-silenced lines (as in Fig. 1) were analyzed with the indicated antibodies. B, Total protein samples from rosette leaves of 4-week-old wild-type and siVPS45-10d plants were analyzed with an anti-VSR antibody. C, Left panels, Wild-type and siVPS45-10d seedlings were grown in vitro for 2 weeks in liquid Murashige and Skoog medium supplemented with 1% Suc and homogenized in 100 mm Tris, pH 8, 5 mm EDTA, and complete protease inhibitor cocktail. Pellet (P16) and soluble (S16) fractions after centrifugation at 16,000g were analyzed with an anti-VSR antibody. Right panels, Pellet (P8) and soluble (S8) fractions after centrifugation at 8,000g were analyzed with the indicated antibodies. D, Microsomes were prepared from wild-type and siVPS45-1a plants grown in liquid for 20 d, separated in a step Suc gradient as described (Bassham and Raikhel, 1998), and analyzed with the indicated antibodies. Estimated in-gel sizes of the bands detected by the antibodies: AtVPS45, 66 kD; VSR, 80 kD; SYP21, 36 kD; SYP41, 39 kD; VCL1, 100 kD; VPS33, 70 kD. E, Wild-type, siVPS45-1a, and siVPS45-10d plants transgenic for a GFP-VSR3 construct (Miao et al., 2008) were obtained by crossing to a stable transgenic GFP-VSR3 line. Roots were incubated in liquid Murashige and Skoog medium with 50 μm wortmannin (Wortmannin) or a similar amount of dimethyl sulfoxide used as a solvent (Control), and epidermal cells of the root tip were imaged 5 h later. Green signal indicates VSR3-GFP, and red signal indicates propidium iodide. Bars = 10 μm. Wt, Wild type; si10d, siVPS45-10d; si1a, siVPS45-1a.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ahmed SU, Rojo E, Kovaleva V, Venkataraman S, Dombrowski JE, Matsuoka K, Raikhel NV (2000) The plant vacuolar sorting receptor AtELP is involved in transport of NH(2)-terminal propeptide-containing vacuolar proteins in Arabidopsis thaliana. J Cell Biol 149 1335–1344 - PMC - PubMed
    1. Andreasson E, Bolt Jorgensen L, Hoglund AS, Rask L, Meijer J (2001) Different myrosinase and idioblast distribution in Arabidopsis and Brassica napus. Plant Physiol 127 1750–1763 - PMC - PubMed
    1. Bassham DC, Raikhel NV (1998) An Arabidopsis VPS45p homolog implicated in protein transport to the vacuole. Plant Physiol 117 407–415 - PMC - PubMed
    1. Bassham DC, Sanderfoot AA, Kovaleva V, Zheng H, Raikhel NV (2000) AtVPS45 complex formation at the trans-Golgi network. Mol Biol Cell 11 2251–2265 - PMC - PubMed
    1. Bryant NJ, James DE (2001) Vps45p stabilizes the syntaxin homologue Tlg2p and positively regulates SNARE complex formation. EMBO J 20 3380–3388 - PMC - PubMed

Publication types

MeSH terms