Functional Identification of Sorting Receptors Involved in Trafficking of Soluble Lytic Vacuolar Proteins in Vegetative Cells of Arabidopsis

Lee, Yong-Jik; Jang, Mihue; Song, Kyungyoung; Kang, Hyangju; Lee, Myoung Hui; Lee, Dong Hoon; Zouhar, Jan; Rojo, Enrique; Sohn, Eun Ju; Hwang, Inhwan

doi:10.1104/pp.112.210914

Cited by 25 publications

(39 citation statements)

References 55 publications

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“…For instance, the expression of mutants of the sorting nexins (which locate to the TGN) inhibits vacuolar protein transport and leads to the accumulation of vacuolar cargo at the ER (Niemes et al, 2010). Vacuolar proteins also have been shown to accumulate in the ER in vsr1 and vsr3 double mutants (Lee et al, 2013). Concerning adaptins, certain vacuolar (Arabidopsis aleurin-like protease) and secretory (invertase) cargos have been shown to accumulate at the ER in a mutant of the m-subunit of the AP-1 complex, which is involved in late secretory and vacuolar traffic (Park et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

Sorting Motifs Involved in the Trafficking and Localization of the PIN1 Auxin Efflux Carrier

et al. 2016

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In contrast with the wealth of recent reports about the function of m-adaptins and clathrin adaptor protein (AP) complexes, there is very little information about the motifs that determine the sorting of membrane proteins within clathrin-coated vesicles in plants. Here, we investigated putative sorting signals in the large cytosolic loop of the Arabidopsis (Arabidopsis thaliana) PIN-FORMED1 (PIN1) auxin transporter, which are involved in binding m-adaptins and thus in PIN1 trafficking and localization. We found that Phe-165 and Tyr-280, Tyr-328, and Tyr-394 are involved in the binding of different m-adaptins in vitro. However, only Phe-165, which binds mA(m2)-and mD(m3)-adaptin, was found to be essential for PIN1 trafficking and localization in vivo. The PIN1:GFP-F165A mutant showed reduced endocytosis but also localized to intracellular structures containing several layers of membranes and endoplasmic reticulum (ER) markers, suggesting that they correspond to ER or ER-derived membranes. While PIN1:GFP localized normally in a mA (m2)-adaptin mutant, it accumulated in big intracellular structures containing LysoTracker in a mD (m3)-adaptin mutant, consistent with previous results obtained with mutants of other subunits of the AP-3 complex. Our data suggest that Phe-165, through the binding of mA (m2)-and mD (m3)-adaptin, is important for PIN1 endocytosis and for PIN1 trafficking along the secretory pathway, respectively.

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Section: Discussionmentioning

confidence: 99%

Sorting Motifs Involved in the Trafficking and Localization of the PIN1 Auxin Efflux Carrier

et al. 2016

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“…In addition, two cargo recognition vacuolar sorting receptors, (VSR1 and VSR4) were identified with RFP-RABF2b/ARA7 enrichment only, and VSR3 was identified with YFP-VAMP711 baits. These VSRs are involved with sorting cargo from the TGN to the lytic vacuole and to the seed protein storage vacuole (103).…”

Section: The Yfp-got1 Enrichment Is Consistent With Previously Publismentioning

confidence: 99%

Identification of Regulatory and Cargo Proteins of Endosomal and Secretory Pathways in Arabidopsis thaliana by Proteomic Dissection*

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Tomé

et al. 2015

Molecular & Cellular Proteomics

100

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The cell's endomembranes comprise an intricate, highly dynamic and well-organized system. In plants, the proteins that regulate function of the various endomembrane compartments and their cargo remain largely unknown. Our aim was to dissect subcellular trafficking routes by enriching for partially overlapping subpopulations of endosomal proteomes associated with endomembrane markers. We selected RABD2a/ARA5, RABF2b/ARA7, RABF1/ARA6, and RABG3f as markers for combinations of the Golgi, trans-Golgi network (TGN), early endosomes (EE), secretory vesicles, late endosomes (LE), multivesicular bodies (MVB), and the tonoplast. As comparisons we used Golgi transport 1 (GOT1), which localizes to the Golgi, clathrin light chain 2 (CLC2) labeling clathrincoated vesicles and pits and the vesicle-associated membrane protein 711 (VAMP711) present at the tonoplast. We developed an easy-to-use method by refining published protocols based on affinity purification of fluorescent fusion constructs to these seven subcellular marker proteins in Arabidopsis thaliana seedlings. We present a total of 433 proteins, only five of which were shared among all enrichments, while many proteins were common between endomembrane compartments of the same trafficking route. Approximately half, 251 proteins, were assigned to one enrichment only. Our dataset contains known regulators of endosome functions including small GTPases, SNAREs, and tethering complexes. We identify known cargo proteins such as PIN3, PEN3, CESA, and the recently defined TPLATE complex. The subcellular localization of two GTPase regulators predicted from our enrichments was validated using live-cell imaging. This is the first proteomic dataset to discriminate between such highly overlapping endomembrane compartments in plants and can be used as a general proteomic resource to predict the localization of proteins and identify the components of regulatory complexes and provides a useful tool for the identification of new protein markers of the endomembrane system. Molecular & Cellular

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“…This idea is similar to the concept proposed by the M&M model (Li and Teng, 2013). To test this idea, we first introduced two critical sequence motifs, FNGLK and FP/RK, of the RbcS TP into the N-terminal 80-amino acid segment (CPY80) of carboxypeptidase Y (CPY), a vacuolar protein that is targeted to the ER via a signal recognition particle-dependent pathway and finally transported to the vacuole via vesicle trafficking through the Golgi apparatus and prevacuolar compartment (Lee et al, 2013b). As expected, CPY delivered GFP to the central vacuole, but not to the chloroplast (Fig.…”

Section: An Efficient Syntp Can Be Generated From An Unrelated Sequenmentioning

confidence: 99%

“…The sequences of the primers used to construct reporter constructs are shown in Supplemental Table S1. To generate CPY[1-95]:GFP, CPY:GFP (Lee et al, 2013b) was digested with XbaI/SalI, and the digested DNA fragment corresponding to CPY[1-95] was ligated to an expression vector digested with the same restriction endonucleases. To generate SynTPs, we performed four successive PCR reactions with four forward primers (in the order of Syn-1, Syn-2, Syn-3, and Syn-4; Supplemental Table S1) and nosT-B primer (reverse primer) using RbcS-nt:GFP as template.…”

Section: Construction Of Reporter Constructsmentioning

confidence: 99%

Sequence Motifs in Transit Peptides Act as Independent Functional Units and Can Be Transferred to New Sequence Contexts

et al. 2015

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A large number of nuclear-encoded proteins are imported into chloroplasts after they are translated in the cytosol. Import is mediated by transit peptides (TPs) at the N termini of these proteins. TPs contain many small motifs, each of which is critical for a specific step in the process of chloroplast protein import; however, it remains unknown how these motifs are organized to give rise to TPs with diverse sequences. In this study, we generated various hybrid TPs by swapping domains between Rubisco small subunit (RbcS) and chlorophyll a/b-binding protein, which have highly divergent sequences, and examined the abilities of the resultant TPs to deliver proteins into chloroplasts. Subsequently, we compared the functionality of sequence motifs in the hybrid TPs with those of wild-type TPs. The sequence motifs in the hybrid TPs exhibited three different modes of functionality, depending on their domain composition, as follows: active in both wild-type and hybrid TPs, active in wild-type TPs but inactive in hybrid TPs, and inactive in wild-type TPs but active in hybrid TPs. Moreover, synthetic TPs, in which only three critical motifs from RbcS or chlorophyll a/bbinding protein TPs were incorporated into an unrelated sequence, were able to deliver clients to chloroplasts with a comparable efficiency to RbcS TP. Based on these results, we propose that diverse sequence motifs in TPs are independent functional units that interact with specific translocon components at various steps during protein import and can be transferred to new sequence contexts.

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Functional Identification of Sorting Receptors Involved in Trafficking of Soluble Lytic Vacuolar Proteins in Vegetative Cells of Arabidopsis

Cited by 25 publications

References 55 publications

Sorting Motifs Involved in the Trafficking and Localization of the PIN1 Auxin Efflux Carrier

Sorting Motifs Involved in the Trafficking and Localization of the PIN1 Auxin Efflux Carrier

Identification of Regulatory and Cargo Proteins of Endosomal and Secretory Pathways in Arabidopsis thaliana by Proteomic Dissection*

Sequence Motifs in Transit Peptides Act as Independent Functional Units and Can Be Transferred to New Sequence Contexts

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