A small molecule inhibitor partitions two distinct pathways for trafficking of tonoplast intrinsic proteins in Arabidopsis

PLoS One. 2012;7(9):e44735. doi: 10.1371/journal.pone.0044735. Epub 2012 Sep 5.

Abstract

Tonoplast intrinsic proteins (TIPs) facilitate the membrane transport of water and other small molecules across the plant vacuolar membrane, and members of this family are expressed in specific developmental stages and tissue types. Delivery of TIP proteins to the tonoplast is thought to occur by vesicle-mediated traffic from the endoplasmic reticulum to the vacuole, and at least two pathways have been proposed, one that is Golgi-dependent and another that is Golgi-independent. However, the mechanisms for trafficking of vacuolar membrane proteins to the tonoplast remain poorly understood. Here we describe a chemical genetic approach to unravel the mechanisms of TIP protein targeting to the vacuole in Arabidopsis seedlings. We show that members of the TIP family are targeted to the vacuole via at least two distinct pathways, and we characterize the bioactivity of a novel inhibitor that can differentiate between them. We demonstrate that, unlike for TIP1;1, trafficking of markers for TIP3;1 and TIP2;1 is insensitive to Brefeldin A in Arabidopsis hypocotyls. Using a chemical inhibitor that may target this BFA-insensitive pathway for membrane proteins, we show that inhibition of this pathway results in impaired root hair growth and enhanced vacuolar targeting of the auxin efflux carrier PIN2 in the dark. Our results indicate that the vacuolar targeting of PIN2 and the BFA-insensitive pathway for tonoplast proteins may be mediated in part by common mechanisms.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Acyltransferases / genetics
  • Acyltransferases / physiology*
  • Aquaporins / genetics
  • Aquaporins / physiology*
  • Arabidopsis / drug effects*
  • Arabidopsis / metabolism*
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism
  • Arabidopsis Proteins / physiology*
  • Brefeldin A / pharmacology
  • Cell Membrane / metabolism
  • Golgi Apparatus / metabolism
  • Green Fluorescent Proteins / metabolism
  • Hypocotyl / metabolism
  • Microscopy, Confocal / methods
  • Models, Biological
  • Promoter Regions, Genetic
  • Protein Transport
  • Seedlings
  • Vacuoles / metabolism

Substances

  • Aquaporins
  • Arabidopsis Proteins
  • TIP2;1 protein, Arabidopsis
  • tonoplast intrinsic protein 3, Arabidopsis
  • Green Fluorescent Proteins
  • Brefeldin A
  • Acyltransferases
  • TIP1 protein, Arabidopsis

Grant support

ERS was supported by the NC State Initiative for Maximizing Student Diversity (IMSD) program and a U.S. Department of Education Graduate Assistance in Areas of National Need (GAANN) Fellowship. This work was supported by start-up funds from North Carolina State University, a grant for the National Science Foundation (IOS-0951616) and North Carolina Space Grant New Investigators Program award to MRP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.