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. 2015 Apr;167(4):1361-73.
doi: 10.1104/pp.114.253377. Epub 2015 Feb 19.

FYVE1 Is Essential for Vacuole Biogenesis and Intracellular Trafficking in Arabidopsis

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Free PMC article

FYVE1 Is Essential for Vacuole Biogenesis and Intracellular Trafficking in Arabidopsis

Cornelia Kolb et al. Plant Physiol. .
Free PMC article

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Abstract

The plant vacuole is a central organelle that is involved in various biological processes throughout the plant life cycle. Elucidating the mechanism of vacuole biogenesis and maintenance is thus the basis for our understanding of these processes. Proper formation of the vacuole has been shown to depend on the intracellular membrane trafficking pathway. Although several mutants with altered vacuole morphology have been characterized in the past, the molecular basis for plant vacuole biogenesis has yet to be fully elucidated. With the aim to identify key factors that are essential for vacuole biogenesis, we performed a forward genetics screen in Arabidopsis (Arabidopsis thaliana) and isolated mutants with altered vacuole morphology. The vacuolar fusion defective1 (vfd1) mutant shows seedling lethality and defects in central vacuole formation. VFD1 encodes a Fab1, YOTB, Vac1, and EEA1 (FYVE) domain-containing protein, FYVE1, that has been implicated in intracellular trafficking. FYVE1 localizes on late endosomes and interacts with Src homology-3 domain-containing proteins. Mutants of FYVE1 are defective in ubiquitin-mediated protein degradation, vacuolar transport, and autophagy. Altogether, our results show that FYVE1 is essential for plant growth and development and place FYVE1 as a key regulator of intracellular trafficking and vacuole biogenesis.

Figures

Figure 1.
Figure 1.
The vfd1(fyve1-2) mutation causes seedling lethality and defects in central vacuole formation. A and B, Phenotypes of vfd1(fyve1-2). A, Seven-day-old seedlings of vfd1(fyve1-2) are shown compared with wild-type (WT) seedlings at the same age. B, Confocal microscopy images of the vacuolar membrane marker GFP-δTIP in 7-d-old wild-type and vfd1(fyve1-2) mutant epidermis cells. C, Schematic presentation of the FYVE1 domain structure. Position of the vfd1 mutation (C415T) leading to a premature stop codon (Q139*) is shown. CC, Coiled-coil domain. Bars = 0.5 mm.
Figure 2.
Figure 2.
The transposon insertion line fyve1-1 shows a similar phenotype as vfd1. A, Schematic presentation of the transposon insertion site in fyve1-1. Gray boxes, Untranslated regions; black boxes, exons; lines, introns. Forward and reverse primer positions used in C are indicated. B, A 7-d-old seedling of fyve1-1 is shown compared with a wild-type (WT) seedling at the same age. C, qRT-PCR analysis of fyve1-1. Expression of FYVE1 in wild-type and fyve1-1 seedlings was analyzed with FYVE1- and ACTIN8 (ACT8)-specific primers. D, Root morphology of fyve1-1. Scanning electron micrographs of wild-type and fyve1-1 root tips. E, Confocal microscopy images of the vacuolar membrane marker GFP-δTIP in 5-d-old wild-type and vfd1(fyve1-2) epidermis cells. Bars = 0.5 mm (B), 100 µm (D), and 10 µm (E).
Figure 3.
Figure 3.
Vacuoles in fyve1-1 show tubular-like interconnected structures. A and C, Vacuole morphology in 2-d-old wild-type (WT; A) and 5-d-old fyve1-1 (C) root epidermis cells visualized by BCECF staining. White boxes, The cells used for surface rendering. DIC, Differential interference contrast image. B and D, Representative three-dimensional surface renderings of wild-type (B) and fyve1-1 (D) vacuoles in root epidermis cells. Views from the front (left) and the side (right) are shown. Bars = 10 µm (A and C) and 5 µm (B and D).
Figure 4.
Figure 4.
fyve1 mutants accumulate ubiquitinated proteins in the membrane fraction. A and B, Immunoblots with an anti-ubiquitin P4D1 antibody from total protein extracts. A, Total extracts of the 7-d-old wild type (WT) and vfd1(fyve1-2) together with two complemented lines of vfd1(fyve1-2) were subjected to immunoblotting with anti-ubiquitin (anti-UB) antibody. CELL DIVISION CYCLE2 (CDC2) was used as a loading control. B, Seven-day-old fyve1-1 mutants compared with wild-type seedlings of the same age were analyzed with an antiubiquitin immunoblot. CDC2 was used as a loading control. C, Total extracts (S13) of 7-d-old fyve1-1 mutants were fractionated by ultracentrifugation to separate the microsomal (P100) and soluble fraction (S100) and subjected to immunoblotting using an antiubiquitin antibody. Note that the majority of ubiquitinated proteins accumulate in the membrane (P100) fraction. Anti-UDP-glucose pyrophosphorylase (UGPase) and anti-H+-adenosine triphosphatase (ATPase) antibodies were used for controls for the soluble and membrane fractions, respectively. IB, Immunoblot.
Figure 5.
Figure 5.
fyve1-1 mutants are impaired in 12S globulin and 2S albumin processing. A, Seeds of the wild type (WT; left) together with GFP-CT24-expressing fyve1-1 homozygous (middle) or fyve1-1 heterozygous and wild-type seeds (right). Seeds were photographed with a binocular microscope (top) or an epifluorescent microscope (bottom). Genotyping results are shown in Supplemental Figure S3. B, GFP-CT24 in mature embryos of the wild type and fyve1-1. Note that whereas in the wild type, GFP-CT24 signals (green) overlap with the autofluorescence of the seed storage vacuoles (magenta), in fyve1-1, GFP-CT24 is secreted into the intercellular spaces. C, A Coomassie Blue-stained gradient gel of total protein extracts of the wild type and fyve1-1 carrying GFP-CT24. p12S, 12S globulin precursors. D to F, Immunoblots of the same extracts using anti-12S globulin, anti-2S albumin, and anti-GFP antibodies to detect 12S globulin subunits and 12S globulin precursors (D), 2S albumin and 2S albumin precursors (p2S; E), and GFP-CT24 (F). Asterisk indicates an unspecific band. Bar = 1 mm. IB, Immunoblot.
Figure 6.
Figure 6.
fyve1-1 accumulates autophagosome markers. Total extracts from wild-type (WT), fyve1-1, atg7-2, and atg10-1 seedlings grown under long-day conditions were subjected to immunoblotting using an anti-NBR1 and an anti-ATG8 antibody. CDC2 was used as a loading control. IB, Immunoblot.
Figure 7.
Figure 7.
GFP-FYVE1 colocalizes with ARA7. A, Localization of GFP-FYVE1 and mRFP-SYP43 in 5-d-old root epidermis cells. The percentage of GFP-FYVE1 vesicle colocalizing with mRFP-SYP43 is indicated at bottom. Total number of GFP-FYVE1 vesicle counted was n = 257. B and C, Localization of GFP-FYVE1 and mRFP-SYP43 upon 45 min of BFA treatment. Magnification of a BFA body in the area indicated in B is shown in C. Note that mRFP-SYP43, but not GFP-FYVE1, localizes to BFA bodies. D, Localization of GFP-FYVE1 and mCherry-ARA7 in 5-d-old root epidermis cells. The percentage of GFP-FYVE1 vesicle colocalizing with mCherry-ARA7 is indicated at the bottom. The total number of GFP-FYVE1 vesicle counted was n = 253. E and F, Localization of GFP-FYVE1 and mCherry-ARA7 and upon 45 min of WM treatment. Magnification of a WM compartment in the area indicated in E is shown in F. Note that both proteins localize to WM compartments. G, Total extracts (S13) from the wild type (WT) and complementing GFP-FYVE1 line were subjected to ultracentrifugation to separate soluble (S100) and microsomal (P100) fractions. Proteins from each fraction were subjected to immunoblotting using anti-FYVE1. Anti-UGPase and anti-H+-ATPase antibodies were used for controls for soluble and membrane fractions, respectively. IB, Immunoblot.
Figure 8.
Figure 8.
FYVE1 interacts with SH3P2 and SH3P3. YTH analysis of GAD-FYVE1(∆FYVE) with GBD-fused SH3P2 and SH3P3. Transformants were grown on media lacking Leu and Trp (−LW) or Leu, Trp, and His (−LWH) with the indicated amount of 3-amino-1,2,4-triazole (3AT) to test their auxotrophic growth. The expression of all fusion proteins was verified by immunoblotting (Supplemental Fig. S4).
Figure 9.
Figure 9.
FYVE1 colocalizes with SH3P2 on SKD1(EQ)-induced class E compartments. A to C, GFP-SH3P2-expressing plants were crossed with CLC-mKO-expressing (A), mRFP-SYP43-expressing (B), and mCherry-ARA7-expressing (C) lines. Colocalization was analyzed in root epidermis cells of 5-d-old seedlings. The percentage of GFP-SH3P2 vesicle colocalizing with each marker is presented at bottom. Total numbers of GFP-SH3P2 vesicle counted were n = 619 for A, n = 162 for B, and n = 158 for C. D and F, Coexpression analysis of SH3P2-GFP with mCherry-ARA7 in Arabidopsis cell culture-derived protoplasts. Cells in E were treated with WM for 120 min before observation. Note that whereas mCherry-ARA7 accumulates in WM-induced compartments, GFP-SH3P2 does not. Magnification of a WM compartment in the area indicated in E is shown in F. G to I, Coexpression of SH3P2-GFP and mCherry-AMSH3 with SKD1(WT; G) or SKD1(EQ; H) in Arabidopsis cell culture-derived protoplasts. Note that a fraction of GFP-SH3P2 colocalizes with mCherry-AMSH3 in SKD1(EQ)-induced class E compartments. Magnification of a class E compartment in the area indicated in H is shown in I. J and K, Localization of GFP-SH3P2 with TagRFP-FYVE1 upon coexpression of SKD1(EQ) in an Arabidopsis root-derived protoplast. Magnification of a class E compartment in the area indicated in J is shown in K.
Figure 10.
Figure 10.
FYVE1 localizes to LEs, regulates intracellular trafficking and vacuole biogenesis and interacts with SH3P2. FYVE1 function together with SH3P2 or other interactors is essential for endocytic protein degradation, vacuolar protein transport, autophagy, and vacuole biogenesis. Lack of FYVE1 causes seedling lethality, indicating that FYVE1 is essential for plant growth and development. ER, Endoplasmic reticulum.

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