Structural Requirements for Ligand Binding by a Probable Plant Vacuolar Sorting Receptor

Cao, Xiaofeng; Rogers, Sally W.; Butler, Juliet M.; Beevers, Leonard; Rogers, John C.

doi:10.1105/tpc.12.4.493

Cited by 102 publications

(70 citation statements)

References 31 publications

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“…The Vps10p domain involved in ligand recognition may contain two binding sites, one for sequence-specific interactions and the other for unfolded structures (Jørgensen et al, 1999). Structural requirements for VSR PS-1 ligand binding have been published recently (Cao et al, 2000). The authors also found indications for two possible recognition sites in VSR PS-1 , one specific for the NPIR motif and another defined as a "non-NPIR site."…”

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confidence: 60%

Demonstration in Yeast of the Function of BP-80, a Putative Plant Vacuolar Sorting Receptor

et al. 2001

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BP-80, later renamed VSR PS-1 , is a putative receptor involved in sorting proteins such as proaleurain to the lytic vacuole, with its N-terminal domain recognizing the vacuolar sorting determinant. Although all VSR PS-1 characteristics and in vitro binding properties described so far favored its receptor function, this function remained to be demonstrated. Here, we used green fluorescent protein (GFP) as a reporter in a yeast mutant strain defective for its own vacuolar receptor, Vps10p. By expressing VSR PS-1 together with GFP fused to the vacuolar sorting determinant of petunia proaleurain, we were able to efficiently redirect the reporter to the yeast vacuole. VSR PS-1 is ineffective on GFP either alone or when fused with another type of plant vacuolar sorting determinant from a chitinase. The plant VSR PS-1 therefore interacts specifically with the proaleurain vacuolar sorting determinant in vivo, and this interaction leads to the transport of the reporter protein through the yeast secretory pathway to the vacuole. This finding demonstrates VSR PS-1 receptor function but also emphasizes the differences in the spectrum of ligands between Vps10p and its plant equivalent. INTRODUCTIONThe plant secretory system is far less easy to study than its yeast counterpart, mainly due to the lack of an equivalent mutant collection. Plant and yeast cells, in opposition to mammalian cells, do not use the mannose 6-phosphatebased lysosomal sorting tag (Kornfeld, 1992); instead, the sorting information is carried by a peptidic sequence. In yeast, the two vacuolar proteins carboxypeptidase Y (CPY; Johnson et al., 1987) and proteinase A (Klionsky and Emr, 1989) both are produced with an N-terminal propeptide responsible for the vacuolar location of the mature protein. Almost 50 mutants defective for vacuolar protein sorting ( vps ) have allowed the identification of proteins involved in the yeast vacuolar sorting pathway. One of these proteins, Vps10p, plays a central role because it is the vacuolar receptor for CPY (Marcusson et al., 1994). Vps10p also is able to send foreign or malfolded proteins to the vacuole for degradation in a process that is believed to be independent of any sorting signal (Hong et al., 1996).In plants, two main types of vacuolar sorting determinants (VSDs) have been well studied and are believed to correspond to two separate vacuolar sorting pathways. The first type could be defined as a sequence-specific VSD (ssVSD) and has been well studied for barley aleurain (Holwerda et al., 1992) and sweet potato sporamin A (Matsuoka and Nakamura, 1991). For these two examples, the VSD is located within an N-terminal propeptide and contains a conserved tetrapeptide NPIR, the Ile being essential (Matsuoka and Nakamura, 1999). The position of this category of VSD seems to be less important than its sequence specificity, as shown with sporamin A (Koide et al., 1997). The second type of VSD is found in C-terminal propeptides (Ct-VSD); it is rather short with no obvious sequence conservation but needs to be accessibl...

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confidence: 60%

Demonstration in Yeast of the Function of BP-80, a Putative Plant Vacuolar Sorting Receptor

et al. 2001

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“…General methods for construction of recombinant plasmids, characterization of cloned inserts, transformation of tobacco (Nicotiana tabacum) BY-2 cells, maintenance of transgenic tobacco BY-2 cells, and preparation and characterization of antibodies have been described previously Rogers, 1998, 2001;Cao et al, 2000;Jiang et al, 2000;Tse et al, 2004). BY-2 cells were maintained by subculture twice a month on agar plates or every 7 d in liquid cultures at room temperature (21°C-22°C).…”

Section: Methodsmentioning

confidence: 99%

Dynamic Response of Prevacuolar Compartments to Brefeldin A in Plant Cells

Tse

Hillmer

et al. 2006

Plant Physiology

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Little is known about the dynamics and molecular components of plant prevacuolar compartments (PVCs) in the secretory pathway. Using transgenic tobacco (Nicotiana tabacum) Bright-Yellow-2 (BY-2) cells expressing membrane-anchored yellow fluorescent protein (YFP) reporters marking Golgi or PVCs, we have recently demonstrated that PVCs are mobile multivesicular bodies defined by vacuolar sorting receptor proteins. Here, we demonstrate that Golgi and PVCs have different sensitivity in response to brefeldin A (BFA) treatment in living tobacco BY-2 cells. BFA at low concentrations (5-10 mg mL 21 ) induced YFPmarked Golgi stacks to form both endoplasmic reticulum-Golgi hybrid structures and BFA-induced aggregates, but had little effect on YFP-marked PVCs in transgenic BY-2 cells at both confocal and immunogold electron microscopy levels. However, BFA at high concentrations (50-100 mg mL 21 ) caused both YFP-marked Golgi stacks and PVCs to form aggregates in a dose-and timedependent manner. Normal Golgi or PVC signals can be recovered upon removal of BFA from the culture media. Confocal immunofluorescence and immunogold electron microscopy studies with specific organelle markers further demonstrate that the PVC aggregates are distinct, but physically associated, with Golgi aggregates in BFA-treated cells and that PVCs might lose their internal vesicle structures at high BFA concentration. In addition, vacuolar sorting receptor-marked PVCs in root-tip cells of tobacco, pea (Pisum sativum), mung bean (Vigna radiata), and Arabidopsis (Arabidopsis thaliana) upon BFA treatment are also induced to form similar aggregates. Thus, we have demonstrated that the effects of BFA are not limited to endoplasmic reticulum and Golgi, but extend to PVC in the endomembrane system, which might provide a quick tool for distinguishing Golgi from PVC for its identification and characterization, as well as a possible new tool in studying PVC-mediated protein traffic in plant cells.

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“…However, BP-80 also is known to bind to the C-terminal propeptide of 2S albumin, which lacks the NPIR motif (Kirsch et al, 1996). BP-80 is a member of a family of membrane proteins termed vacuolar sorting receptors (VSRs; Paris et al, 1997;Shimada et al, 1997;Hadlington and Denecke 2000;Paris and Neuhaus, 2002), whose function has been confirmed both in vitro (Cao et al, 2000) and in vivo using a yeast expression system (Humair et al, 2001). Targeting determinants also have been identified for proteins sorted into the PSV (Frigerio et al, 1998;Neuhaus and Rogers, 1998).…”

Section: Introductionmentioning

confidence: 99%

Identification of Multivesicular Bodies as Prevacuolar Compartments inNicotiana tabacumBY-2 Cells[W]

et al. 2004

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Little is known about the dynamics and molecular components of plant prevacuolar compartments (PVCs). We have demonstrated recently that vacuolar sorting receptor (VSR) proteins are concentrated on PVCs. In this study, we generated transgenic Nicotiana tabacum (tobacco) BY-2 cell lines expressing two yellow fluorescent protein (YFP)-fusion reporters that mark PVC and Golgi organelles. Both transgenic cell lines exhibited typical punctate YFP signals corresponding to distinct PVC and Golgi organelles because the PVC reporter colocalized with VSR proteins, whereas the Golgi marker colocalized with mannosidase I in confocal immunofluorescence. Brefeldin A induced the YFP-labeled Golgi stacks but not the YFP-marked PVCs to form typical enlarged structures. By contrast, wortmannin caused YFP-labeled PVCs but not YFPlabeled Golgi stacks to vacuolate. VSR antibodies labeled multivesicular bodies (MVBs) on thin sections prepared from highpressure frozen/freeze substituted samples, and the enlarged PVCs also were indentified as MVBs. MVBs were further purified from BY-2 cells and found to contain VSR proteins via immunogold negative staining. Similar to YFP-labeled Golgi stacks, YFP-labeled PVCs are mobile organelles in BY-2 cells. Thus, we have unequivocally identified MVBs as PVCs in N. tabacum BY-2 cells. Uptake studies with the styryl dye FM4-64 strongly indicate that PVCs also lie on the endocytic pathway of BY-2 cells.

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Structural Requirements for Ligand Binding by a Probable Plant Vacuolar Sorting Receptor

Cited by 102 publications

References 31 publications

Demonstration in Yeast of the Function of BP-80, a Putative Plant Vacuolar Sorting Receptor

Demonstration in Yeast of the Function of BP-80, a Putative Plant Vacuolar Sorting Receptor

Dynamic Response of Prevacuolar Compartments to Brefeldin A in Plant Cells

Identification of Multivesicular Bodies as Prevacuolar Compartments inNicotiana tabacumBY-2 Cells[W]

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