AtPAP2 is a tail-anchored protein in the outer membrane of chloroplasts and mitochondria

Abstract

Tail-anchored (TA) proteins possess an N-terminal functional domain and a single transmembrane domain (TMD) at the C-terminus followed by a hydrophilic tail.1 Newly synthesized TA proteins are released from free ribosomes with the C-terminal hydrophobic region inserted into various membranes, such as the endoplasmic reticulum, chloroplast outer envelope, mitochondrial outer membrane and the peroxisomal membrane. 2The functional domain of TA proteins orients to the cytosol and the TMD of TA proteins is inserted into membranes posttranslationally. Sorting of proteins by the C-terminal tail (CT) to their specific intracellular destinations is essential for their functions.3 For instance, overexpression of a C-terminal TMDtruncated AtPAP2 in Arabidopsis abolishes its faster plant growth phenotype. 4 Over 500 proteins in Arabidopsis have been predicted to have TA structures, of which, 130 have had their subcellular localization experimentally confirmed based on either GFP targeting or mass spectrometry.2 Most TA proteins were assigned to the ER and secretory membranes, 27 proteins to mitochondria and 32 proteins to plastids.2,5 These include several isoforms of Tom20 and Tom22 (also known as Tom9) of the mitochondrial outer membrane translocon 6,7 and the GTPase receptors of the outer membrane translocon of plastids, including AtToc33, AtToc34. 5,8Arabidopsis purple acid phosphatase 2 (AtPAP2) is the only plant TA protein shown to be dual-targeted to chloroplasts and mitochondria. 4 It was predicted to carry a putative N-terminal signal peptide, a phosphatase domain and a transmembrane domain (TMD) followed by a short hydrophilic C-terminal tail (CT) (a.a. 614-636) by the TMHMM analysis.4 AtPAP2 was to date, Arabidopsis purple acid phosphatase 2 (AtPAP2) is the only known plant protein that is dual-targeted to chloroplasts and mitochondria by a C-terminal targeting signal. using in vitro organelle import and green fluorescence protein (GFP) localization assays, we showed that AtPAP2 is located on, but not imported across the outer membrane (om) of chloroplasts and mitochondria and exposed its n-terminal enzymatic domain to the cytosol. it was also found that a short stretch of 30 amino acids (a.a.) at the C-terminal region (a.a. 615-644) that contains a stretch of 18 hydrophobic residues, a WYAK motif and 8 hydrophilic residues is sufficient for dual-targeting. mutation of WYAK to WYAE had no effect on dual-targeting ability suggesting that the charge within this flanking region alone is not an important determinant for dual-targeting.AtPAP2 is a tail-anchored protein in the outer membrane of chloroplasts and mitochondria Keywords: purple acid phosphatase, mitochondria, plastid, dual-targeting, outer membrane detected in the membrane fraction using immunoblotting. 4 An in vivo targeting assay using chimeric GFP vectors showed that the C-terminal TMD motif of AtPAP2, but not the predicted N-terminal signal peptide, can direct GFP to both plastids and mitochondria in Arabidopsis PSB-D protoplasts. 4 In transgenic Arabi...

Elongator Protein 3 (Elp3) Lysine Acetyltransferase Is a Tail-anchored Mitochondrial Protein in Toxoplasma gondii

“…These proteins are referred to as tail-anchored (TA) and, while the precise mechanism of mitochondrial membrane insertion is still unclear, mutational analyses have shown that the TMD sequence serves as a mitochondrial targeting signal (33,34). TA proteins are often inserted in the OMM by the C-terminal TMD such that a short C-terminal tail faces the inner membrane space and everything upstream of the TMD resides in the cytosol (35). To determine the orientation of TgElp3 in the mitochondrial membrane, we selectively permeabilized membranes of parasites expressing N-or C-terminally HA-tagged TgElp3 with digitonin prior to IFA analyses.…”

Phosphorylation and Dephosphorylation of the Presequence of Precursor MULTIPLE ORGANELLAR RNA EDITING FACTOR3 during Import into Mitochondria from Arabidopsis

“…By contrast, Arabidopsis plants with all three Tom20 receptor isoforms inactivated show only some reduced growth rate (Lister et al, 2007). In the case of AtPAP2, while overexpression of AtPAP2 results in enhanced growth and higher seed yield (Sun et al, 2012a(Sun et al, , 2012b, sole targeting of AtPAP2 to mitochondria results in early senescence and lower seed yield (Supplemental Fig. S7).…”

“…STY kinases are present in the cytosol, and AtPAP2 is present on the outer membranes of both organelles (Martin et al, 2006;Sun et al, 2012a). In vivo BiFC assays showed that pMORF3/5 interacted with AtPAP2 on mitochondria but not with AtPAP2 on chloroplasts (Fig.…”

“…The C-terminal transmembrane domain of PAP2 anchors AtPAP2 to the outer membrane of chloroplasts and mitochondria (Sun et al, 2012a). AtPAP2 was highly expressed in mitochondria of an Arabidopsis overexpressing AtPAP2 line (OE7) but was not expressed in the pap2 transfer DNA (T-DNA) line (Supplemental Fig.…”

“…Previously, we identified an outer mitochondrial membrane protein, called PURPLE ACID PHOSPHA-TASE2 (AtPAP2), that is also dually targeted to the outer envelope in chloroplasts (Sun et al, 2012a(Sun et al, , 2012b. Similar to Toc33/34 and Tom20, AtPAP2 is anchored on the outer membranes by a C-terminal transmembrane motif.…”