Abstract

Membrane trafficking is essential in eukaryotic cells as a delivery system for newly synthesized proteins from the endoplasmic reticulum (ER) to reach the plasma membrane (PM) or the tonoplast via intermediate endomembrane compartments. The selective transport of macromolecules between different compartments of the endomembrane system is mediated by small vesicles via adaptor complexes (AP-1-4). The ? subunit of AP complexes is devoted to cargo protein selection via a specific and well characterized interaction with a tyrosin-sorting signal (YXX?, where ? is a bulky hydrophobic residue and X is any amino acid). In plant systems, ER and PM provide membrane continuity between cells through the connections made by plasmodesmata (PD). Virus movement, which requires passage of macromolecules through PD connections, is mediated by one or more virus-encoded MPs with the help of the host cytoskeleton and/or endomembranes. The MP encoded by Cauliflower mosaic virus (CaMV) forms tubules guiding encapsidated virus particle cell-cell transport via an indirect MP-virion interaction. CaMV MP does not require an intact cytoskeleton for both PM-targeting and tubule formation. However, how this and the other tubule-forming MPs targets the PM and form tubules remains to be elucidated. In this study, we examined the three tyrosine-sorting motifs in CaMV MP and showed that each of them interacts directly with subunit ? of an Arabidopsis AP-complex. Fluorophore-tagged MP is incorporated into vesicles labeled with the endocytic tracer FM4-64 and the pharmacological interference of tyrphostin A23, an inhibitor of endocytosis, confirmed that MP traffics in the endocytic pathway. Mutations in the three endocytosis domains revert in the viral context suggesting that vesicle carrier activity is essential for CaMV viability. In our system, mutation of the three tyr-signals blocks internalization of the protein from the plasma membrane to early endosomes, PD localization and tubule formation, but does not prevent targeting of newly synthesized MP to the plasma membrane, at least in the early stages of infection. The evidence we provide that upon mutation of all three YXX?-signals MP can efficiently interact with PDLP1, a PD protein involved in assembly of CaMV MP into tubules, indicates that this MP mutant is competent to form tubules and its failure to accumulate in PD and to form tubules more probably depends on the inability to target PDs. This suggests that after targeting the plasma membrane (via as yet unknown strategy), MP might use a recycling pathway for specific targeting of PD via constitutive cycling between EE and PD. As constitutive cycling of plasma membrane proteins is blocked by BFA, recycling of MP is supported here by the demonstration that formation of foci (and tubules) is inhibited upon treatment of protoplasts with BFA. Tyrosine-signals can interact with several ?-adaptins and help the same protein to traffic in different compartments of the end

Autore Pugliese

• Stavolone Livia

Tutti gli autori

• Carluccio AV.; Zicca S.; Stavolone L.

Titolo volume/Rivista

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Anno di pubblicazione

2013

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ISBN

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Numero di citazioni Wos

Nessuna citazione

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Numero di citazioni Scopus

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Settori ERC

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Codici ASJC

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