Targeting of a Nicotiana plumbaginifolia H⁺-ATPase to the Plasma Membrane Is Not by Default and Requires Cytosolic Structural Determinants

Abstract

The structural determinants involved in the targeting of multitransmembrane-span proteins to the plasma membrane (PM) remain poorly understood. The plasma membrane H þ -ATPase (PMA) from Nicotiana plumbaginifolia, a well-characterized 10 transmembrane-span enzyme, was used as a model to identify structural elements essential for targeting to the PM. When PMA2 and PMA4, representatives of the two main PMA subfamilies, were fused to green fluorescent protein (GFP), the chimeras were shown to be still functional and to be correctly and rapidly targeted to the PM in transgenic tobacco. By contrast, chimeric proteins containing various combinations of PMA transmembrane spanning domains accumulated in the Golgi apparatus and not in the PM and displayed slow traffic properties through the secretory pathway. Individual deletion of three of the four cytosolic domains did not prevent PM targeting, but deletion of the large loop or of its nucleotide binding domain resulted in GFP fluorescence accumulating exclusively in the endoplasmic reticulum. The results show that, at least for this polytopic protein, the PM is not the default pathway and that, in contrast with single-pass membrane proteins, cytosolic structural determinants are required for correct targeting.

Molecular Genetic Evidence for the Role of SGT1 in the Intramolecular Complementation of Bs2 Protein Activity in Nicotiana benthamiana

“…Bs2:HA and the cytoplasmically localized NptII and HSP90 (Milioni and Hatzopoulos, 1997) proteins remained in the supernatant after the high-speed spin used to pellet the plant membranes in plants inoculated with buffer alone (Figure 4). Conversely, a plasma membrane-localized H-ATPase (Lefebvre et al, 2004) was concentrated in the pellet after the high-speed spin.…”

Insights into the Role of Specific Lipids in the Formation and Delivery of Lipid Microdomains to the Plasma Membrane of Plant Cells

“…A, The DRM marker proteins PMA2, PIP1, and remorin (Mongrand et al, 2004;Borner et al, 2005) were enriched in the DRM fractions as compared to the PM fractions from which they were isolated. The antibodies raised against the ATPase PMA2 (Maudoux et al, 2000) may recognize PMA4 as well (Lefebvre et al, 2004). The anti-PIP1 antibody may recognize both the PIP1 and PIP2 families (Santoni et al, 2003).…”

“…To investigate the protein content of PM DRMs, we used antibodies raised against three putative protein markers of DRMs (Mongrand et al, 2004;Borner et al, 2005): the H 1 -ATPase PMA2 (Maudoux et al, 2000;Lefebvre et al, 2004), the aquaporin PIP1 (Santoni et al, 2003;Boursiac et al, 2005), and the remorin (Bariola et al, 2004). Figure 1A shows the enrichment of these markers in PM DRMs compared to the PM.…”

“…Farmer (University of Lausanne, Switzerland). The anti-PMA2 antibody may recognize PMA4 as well (Lefebvre et al, 2004). The anti-PIP1 antibody was raised against a 42-amino acid N-terminal peptide of PIP1-1, and due to sequence homologies, may recognize the PIP1 and PIP2 families (Santoni et al, 2003).…”

Plasma Membrane Localization of Solanum tuberosum Remorin from Group 1, Homolog 3 Is Mediated by Conformational Changes in a Novel C-Terminal Anchor and Required for the Restriction of Potato Virus X Movement

“…Ultrapure RemCA peptides were obtained by de novo peptide synthesis (GL Biochem Shanghai Ltd; www.glschina.com). Western-blot analyses were performed using either anti-REM (Raffaele et al, 2009), anti-Plasma Membrane proton pump ATPase (Lefebvre et al, 2004), anti-PIP (Santoni et al, 2003), antiactin, or commercial anti-GFP (Millipore/Roche) antibodies.…”