The Plant Journal (2008) 53, 29–41
doi: 10.1111/j.1365-313X.2007.03314.x
The GRV2/RME-8 protein of Arabidopsis functions in the late endocytic pathway and is required for vacuolar membrane flow Rebecca A. Silady1,2,†, David W. Ehrhardt2, Karen Jackson3, Christine Faulkner3, Karl Oparka3 and Chris R. Somerville1,2,* Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA, 2 Department of Plant Biology, Carnegie Institution, 260 Panama Street, Stanford, CA 94305, USA, and 3 Institute of Molecular Plant Sciences, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK
1
Received 19 August 2007; accepted 22 August 2007. * For correspondence (fax 650 325 6857; e-mail
[email protected]). † Present address: Department of Plant Breeding and Genetics, Max Planck Institute for Plant Breeding Research, Carl-von-Linne´-Weg 10, 50829 Ko¨ln, Germany.
Summary The gravitropism defective 2 (grv2) mutants of Arabidopsis thaliana were previously characterized as exhibiting shoot agravitropism resulting from mutations in a homolog of the Caenorhabditis elegans RECEPTOR-MEDIATED ENDOCYTOSIS-8 (RME-8) gene, which is required in C. elegans for endocytosis. A fluorescent protein fusion to the GRV2 protein localized to endosomes in transgenic plants, and vacuolar morphology was altered in grv2 mutants. A defect in vacuolar membrane dynamics provides a mechanistic explanation for the gravitropic defect, and may also account for the presence of an enlarged vacuole in early embryos, together with a nutrient requirement during seedling establishment. The GRV2-positive endosomes were sensitive to Wortmannin but not brefeldin A (BFA), consistent with GRV2 operating late in the endocytic pathway, prior to delivery of vesicles to the central vacuole. The specific enlargement of GRV2:YFP structures by Wortmannin, together with biochemical data showing that GRV2 co-fractionates with pre-vacuolar markers such as PEP12/SYP21, leads us to conclude that in plants GRV2/RME-8 functions in vesicle trafficking from the multivesicular body/pre-vacuolar compartment to the lytic vacuole. Keywords: prevacuole, late endosome, vesicle, tonoplast, RME-8, endocytosis.
Introduction Shoots and hypocotyls of the gravitropism defective 2 (grv2) mutants of Arabidopsis exhibit a reduced gravity response (Silady et al., 2004). This phenotype appears to be caused by reduced amyloplast sedimentation in the endodermal cells. By contrast amyloplasts in the columella cells of the root sediment properly, and roots of grv2 seedlings respond to gravity. Hypocotyls of grv2 mutants also have a slightly reduced phototropic response (Silady et al., 2004), and a reduction in apical hook maintenance (Silady, 2006), which cannot be accounted for by altered amyloplast sedimentation. GRV2 encodes a protein with sequence similarity to an endocytosis factor, the RME-8 protein from Caenorhabditis elegans, with orthologs in Drosophila and mammalian cells (Silady et al., 2004). RME-8 is required in C. elegans for receptor-mediated endocytosis of yolk protein into growing oocytes. RME-8 is also required for fluid-phase endocytosis ª 2007 The Authors Journal compilation ª 2007 Blackwell Publishing Ltd
of a GFP marker, which is secreted into the body cavity and non-specifically endocytosed by coelomocytes in C. elegans (Zhang et al., 2001). Similarly, mutations in the Drosophila ortholog of RME-8 blocked internalization of the membrane ligand, Boss, into neighboring Sevenless receptor tyrosine kinase-expressing cells, and, in addition, reduced the uptake of endocytosis tracers. Mutations in RME-8 also enhance the rough eye phenotype of a GTP hydrolysis-defective dynamin mutant in Drosophila (Chang et al., 2004). The mechanistic bases for the endocytosis-defective phenotypes of the rme-8 mutants are not known. GRV2 and RME-8 are large proteins of 259 and 277 kDa, respectively, with four IWN repeats and a J-domain. RME-8 localizes to the limiting membrane of large endosomes in the macrophage-like coelomocytes of C. elegans (Zhang et al., 2001), but does not have any predicted or known membrane-spanning domains. Co-localization studies in 29
30 Rebecca A. Silady et al. Drosophila indicated that RME-8 co-localizes with Clc, Rab5 and Rab7, which are markers for clathrin-containing organelles, early endosomes and late endosomes, respectively (Chang et al., 2004). RME-8 interacts with Hsc70-4 via the J-domain in Drosophila and mammalian cells, and may play a role as a co-chaperone in the uncoating of clathrin-coated vesicles (Chang et al., 2004; Girard et al., 2005). Recently, the GRV2 ortholog of tobacco has been found to interact with the TGB2 movement protein (MP) of potato mop-top virus (Haupt et al., 2005). Haupt et al. showed that following the targeting of the viral genome to plasmodesmata, two of the viral MPs (TGB1 and TGB2) were recycled back to the cell interior using an endocytic pathway. The direct interaction of a viral MP with GRV2 is consistent with the view that some viruses may have hijacked components of the endocytic recycling machinery, for recycling to and from plasmodesmata (Haupt et al., 2005). Endocytosed vesicles are transported from the plasma membrane to an endosome either by fusing with an existing endosome or by maturing into an endosome. Endocytosed material is then either cycled back to the plasma membrane or transported via the pre-vacuolar compartment (PVC) to either the vacuole or the trans-Golgi network (TGN) (Lam et al., 2005). Adding to the complexity of vesicle transport, newly synthesized proteins destined for the vacuole are transported from the TGN either directly via the PVC, or are first secreted to the plasma membrane and then endocytosed and transported via the PVC to the vacuole. This results in an overlap between biosynthesis pathways and endocytosis. Indeed, the PVC contains proteins from both pathways (Neuhaus and Paris, 2005). Additional gravitropism mutants, sgr2, sgr3 and zig (zig zag)/sgr4, also show membrane-related defects. SGR2 encodes a putative phospholipase A1, SGR3 encodes the vacuolar t-SNARE AtVAM3 and ZIG/SGR4 encodes the vacuolar v-SNARE AtVTI11 (Kato et al., 2002; Morita et al., 2002). Mutations in these genes are thought to primarily affect the tonoplast, causing alterations in vacuole morphology and dynamics (Morita et al., 2002; Yano et al., 2003), which in turn disrupts amyloplast sedimentation, resulting in the observed gravitropic defects. In this report we describe additional phenotypic effects of grv2 mutations on embryo development and seedling establishment. We also show that GRV2 localizes to endosomes that are sensitive to the drug Wortmannin. Membranes labeled with a tonoplast GFP marker protein form aggregates in the hypocotyls of grv2 etiolated seedlings. These observations complement those in a recent study in which mutations in GVR2, called katamari2 (kam2), were independently isolated on the basis of a direct microscopic screen for altered endomembranes (Tamura et al., 2007). Together, these observations indicate a role for GRV2 in vesicle trafficking to the vacuole, and provide insights into the mechanistic basis for the various mutant phenotypes.
Results The grv2-1 mutant exhibits a defect in embryogenesis Embryogenesis in Arabidopsis normally follows a highly uniform pattern (Jurgens and Mayer, 1994; Mansfield and Briarty, 1991). The first cell division of grv2-1 zygotes follows the same pattern as wild type, resulting in a small apical cell and an elongated basal cell (Figure 1a,g). However, the next two rounds of cell division in the grv2-1 mutant deviate from the wild-type pattern. Rather than dividing through the center of the cell, the newly formed cell wall is shifted to one side resulting in the size of one cell being much greater than the other (Figure 1b,h). This large cell persists until the heart stage (Figure 1e). Despite the abnormal early cell divisions, grv2-1 embryos continue to develop and eventually assume a relatively normal appearance. The main defect observed at the torpedo stage was a low frequency of embryos with three cotyledons (data not shown). By the torpedo stage the enlarged cells were no longer observed (Figure 1f,l). The grv2-1 allele, which was identified in the Landsberg erecta (Ler) background, is not fully penetrant for the embryo phenotype: an average of 77% of homozygous grv2-1 embryos exhibit the large cell. By contrast, mutant alleles in the Columbia (Col) ecotype, grv2-2, grv2-3 and grv2-4, did not exhibit this embryo phenotype (Silady, 2006). Three of five T-DNA lines with insertions in the GRV2 gene (grv2-5, grv2-8 and grv2-9) produced embryos with the enlarged cell, but at a much lower penetrance than the grv2-1 line (Table 1). The GRV2 gene complemented the embryo phenotype of the grv2-1 mutant, indicating that the defects in embryogenesis are caused by the grv2-1 mutation (Silady, 2006). Interallelic crosses between the grv2-1 allele and the grv2-2, grv2-3 and grv2-4 alleles did not result in the expected 3:1 ratio between wild-type embryos and mutant embryos in the resulting F2 populations. Rather, there was a statistically significant excess of mutant embryos (Table 2). Thus, the grv2-1 allele has a residual activity that may eventually be useful in understanding the function of the protein. The grv2-1 allele creates a premature stop codon resulting in an 85-amino-acid truncation, whereas the grv2-2, grv2-3 and grv2-4 mutants are c-ray and fast-neutron alleles resulting in deletions and insertions (Silady, 2006). Viability of the gvr2 mutants When germinated and grown in continuous light on agar plates without sucrose 84–98% of grv2 mutant seedlings arrested, compared with 7–36% of wild-type seedlings (Figure 2a,c, Table 3). By contrast, when grown on agar plates with 1% sucrose only 4% of the mutant seedlings arrested compared with 0% of wild-type seedlings.
ª 2007 The Authors Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2008), 53, 29–41
The GRV2 protein of Arabidopsis 31
(a)
(b)
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(i)
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(k)
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Figure 1. Differential interference microscopy of cleared embryos at sequential developmental stages. One-cell (a, g), quadrant (b, h), dermatogen (c, i), globular (d, j), heart (e, k) and torpedo (f, l) stages. (a–f) grv2-1 mutants. (g–h) Ler wild type. Arrows indicate the enlarged cells; asterisks mark the ends of the cell plate. Scale bars: (a–e) and (g–k), 10 lm; (f–l), 20 lm.
Table 1 Penetrance of the grv2 embryo phenotype in selfed progeny of heterozygous GRV2 T-DNA insertion lines
Allele
T-DNA line
Ecotype
Location of insertion
Penetrance of grv2 embryo phenotype in self-progeny of heterozygous plantsa
grv2-1 grv2-5 grv2-6 grv2-7 grv2-8 grv2-9
EMS allele GT1669 (Sundaresan et al., 1995) 519_H08 (Sessions et al., 2002) 712_G08 (Sessions et al., 2002) 905_G11 (Sessions et al., 2002) Salk 067162 (Alonso et al., 2003)
Ler Ler Col Col Col Col
Stop codon in exon 20 (Silady et al., 2004) Exon 1 (Silady et al., 2004) Exon 15 (Silady, 2006) Exon 7 (Silady, 2006) Intron 10 (Silady, 2006) Intron 13 (Silady et al., 2004)
19%, n = 315 3%, n = 458 0%, n > 90 0%, n > 50 6%, n = 17 9%, n = 159
a P-values from two-tailed Fisher exact tests are 0.4040, 0.0007 and 1 for pair-wise comparisons between GT1669 and 905_G11, GT1669 and Salk 067162, and 905_G11 and Salk 067162, respectively. Similarly, P-values were 1.4 · 10)13, 0.33, and 0.0032 for pair-wise comparisons between grv21 and GT1669, grv2-1 and 905_G11, and grv2-1 and Salk 067162, respectively. n: Number of embryos screened.
The mutant seedlings arrested in the absence of sucrose approximately 3 days after germination. The proportion of arrested seedlings and the morphology of arrested seedlings were indistinguishable among the various grv2 alleles. At the time of arrest the root had elongated to an average length of 1.11 0.1 mm and the cotyledons were green, but the first true leaves had not appeared (Figure 2a). Seedlings that were transferred from plates without sucrose to plates containing sucrose within 6 days of germination recovered and developed into normal seedlings. Seedlings grown on 1% sorbitol also arrested, whereas seedlings grown on 1%
glucose did not, indicating that the sucrose is necessary as a nutrient source rather than as an osmoticum. Subcellular localization of GRV2 To detect GRV2 protein in biochemical studies, an antibody was raised to a 67-kDa region of the 7th exon of GRV2. The antibody recognized a protein of the expected size of 277 kDa on Western blots of protein extracts from wild-type leaves and flowers, but did not recognize a protein of the expected size in protein extracts from grv2-1, grv2-2, grv2-3 or grv2-4
ª 2007 The Authors Journal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2008), 53, 29–41
32 Rebecca A. Silady et al. Table 2 The grv2-1 embryo phenotype is dominant to other alleles in F1 individuals from interallelic crosses
Cross
Phenotypically v2 test Phenotypically a mutant embryos wild-type embryos (P-value)b
grv2-1/GRV2+ self grv2-1/grv2-2 self grv2-1/grv2-3 self grv2-1/grv2-4 self grv2-3/grv2-1 self
61 (19%) 140 (40%) 120 (42%) 176 (37%) 121 (42%)
254 207 163 301 170
