The Destination for Single-Pass Membrane Proteins Is Influenced Markedly by the Length of the Hydrophobic Domain

Abstract

The tonoplast was proposed as a default destination of membrane-bound proteins without specific targeting signals. To investigate the nature of this targeting, we created type I fusion proteins with green fluorescent protein followed by the transmembrane domain of the human lysosomal protein LAMP1. We varied the length of the transmembrane domain from 23 to either 20 or 17 amino acids by deletion within the hydrophobic domain. The resulting chimeras, called TM23, TM20, and TM17, were expressed either transiently or stably in tobacco. TM23 clearly accumulated in the plasmalemma, as confirmed by immunoelectron microscopy. In contrast, TM17 clearly was retained in the endoplasmic reticulum, and TM20 accumulated in small mobile structures. The nature of the TM20-labeled compartments was investigated by coexpression with a marker localized mainly in the Golgi apparatus, AtERD2, fused to a yellow fluorescent protein. The strict colocalization of both fluorescent proteins indicated that TM20 accumulated in the Golgi apparatus. To further test the default destination of type I membrane proteins, green fluorescent protein was fused to the 19-amino acid transmembrane domain of the plant vacuolar sorting receptor BP-80. The resulting chimera also accumulated in the Golgi instead of in post-Golgi compartments, where native BP-80 localized. Additionally, when the transmembrane domain of BP-80 was lengthened to 22 amino acids, the reporter escaped the Golgi and accumulated in the plasma membrane. Thus, the tonoplast apparently is not a favored default destination for type I membrane proteins in plants. Moreover, the target membrane where the chimera concentrates is not unique and depends at least in part on the length of the membrane-spanning domain. INTRODUCTIONThe sorting of integral proteins in plants is not well understood. Nevertheless, it is accepted that peptidic signals exposed in the cytosol are responsible for targeting to the correct subcellular location in plant cells. This signal-mediated sorting is opposed to a default transport that is believed to happen when no signal is present on a protein.Although the default destination within the secretory pathway for a soluble protein is secretion, the default membrane is unclear. The most informative results about the location where membrane proteins would accumulate by default were provided by a study of ␣ -TIP (Höfte and Chrispeels, 1992). In this experiment, the last 48 amino acids of ␣ -TIP, which contains the sixth transmembrane domain, were sufficient to target a reporter protein to the tonoplast. Because the deletion of the cytosolic C-terminal 15 amino acids from the ␣ -TIP sequence did not prevent the tonoplast accumulation of the truncated protein, the authors indirectly deduced a role for the sixth membrane-spanning domain. Either this ␣ -TIP transmembrane domain would be sufficient for vacuolar location or the tonoplast would be the default destination for membrane proteins.More recently, the same sixth transmembrane domain of ␣ -TIP was used in a chimeric c...

Characterization of Four Lectin-Like Receptor Kinases Expressed in Roots of Medicago truncatula. Structure, Location, Regulation of Expression, and Potential Role in the Symbiosis with Sinorhizobium meliloti

“…In studies in yeast (see Rayner and Pelham, 1997) and more recently in plants (Brandizzi et al, 2002), the length of the TM helix has been shown to play a major role in protein sorting because the helix needs to be at least 23 amino acids to span and to permit sorting to the PM. The predicted TM helices of MtLecRK1;1 and MtLecRK7;2 are 23 amino acids in length, thus fulfilling this sorting requirement.…”

Dual Fatty Acyl Modification Determines the Localization and Plasma Membrane Targeting of CBL/CIPK Ca2+ Signaling Complexes in Arabidopsis

“…As a control for the efficiency of the BFA treatment, we analyzed the BFAsensitive localization of TM23:GFP, a modified GFP containing a transmembrane domain (Brandizzi et al, 2002).…”

De Novo Formation of Plant Endoplasmic Reticulum Export Sites Is Membrane Cargo Induced and Signal Mediated

“…Of particular use for our present investigation is TMcCCASP, a chimeric type I protein (Hanton et al, 2005b) consisting of a form of GFP carrying a signal peptide for entry into the ER and an N-glycosylation site (Batoko et al, 2000) fused to a 17-amino acid transmembrane domain, the short length of which restricts type I proteins to the ER (Brandizzi et al, 2002a). Adjacent to the transmembrane domain are 78 amino acids of the cytosolic domain of CASP (Hanton et al, 2005b;Renna et al, 2005), containing a functional diacidic ER export motif that is dominant over the properties of the transmembrane domain and permits Figure 1.…”