The N-Terminal Hydrophobic Region of the Mature Phosphate Translocator Is Sufficient for Targeting to the Chloroplast Inner Envelope Membrane

The triose phosphate 3-phosphoglycerate phosphate translocator (TPT) is a chloroplast envelope inner membrane protein whose transit peptide has structural properties typical of a mitochondrial presequence. To study the TPT transit peptide in more detail, we constructed two chimeric genes encompassing the TPT transit peptide and either 5 or 23 amino-terminal residues of the mature TPT, both linked to the reporter chloramphenicol acetyltransferase (cat) gene. The precursors were synthesized in vitro and translocated to and processed in purified plant mitochondria. However, this import was not specific since both precursors were also imported into isolated chloroplasts. To extend this analysis in vivo, the chimeric genes were introduced into tobacco by genetic transformation. Analysis of CAT distribution in subcellular fractions of transgenic plants did not confirm the data obtained in vitro. With the construct retaining only 5 residues of the mature TPT, CAT was found in the cytosolic fraction. Extension of the TPT transit peptide to 23 residues of the mature TPT allowed specific import and processing of CAT into chloroplasts. These results indicate that, despite its unusual structure, the TPT transit peptide is able to target a passenger protein specifically into chloroplasts, provided that NH 2 -terminal residues of the mature TPT are still present. The discrepancy between the in vitro and in vivo data suggests that the translocation machinery is more stringent in the latter case and that sorting of proteins might not be addressed adequately by in vitro experiments.

Section: The Tpt Transit Peptide Addresses a Reporter Protein Intosupporting

confidence: 87%

Section: Fig 2 Import Of Tpt5-cat and Tpt23-cat Into Isolated Chlorsupporting

confidence: 53%

mentioning

confidence: 99%

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Different in Vitro and in Vivo Targeting Properties of the Transit Peptide of a Chloroplast Envelope Inner Membrane Protein

Silva-Filho¹,

Wieers²,

Flügge³

et al. 1997

“…Moreover, truncation experiments showed that the N-terminal-located extension of the plastid triose-phosphate transporter TPT is not required for proper direction into the inner envelope membrane as the required target information is located in the first hydrophobic domain (56).…”

Section: Discussionmentioning

confidence: 99%

Molecular Identification and Functional Characterization of Arabidopsis thaliana Mitochondrial and Chloroplastic NAD+ Carrier Proteins

Palmieri

Rieder

Ventrella

et al. 2009

157

172

The Arabidopsis thaliana L. genome contains 58 membrane proteins belonging to the mitochondrial carrier family. Two mitochondrial carrier family members, here named AtNDT1 and AtNDT2, exhibit high structural similarities to the mitochondrial nicotinamide adenine dinucleotide (NAD ؉ ) carrierScNDT1 from bakers' yeast. Expression of AtNDT1 or AtNDT2 restores mitochondrial NAD ؉ transport activity in a yeast mutant lacking ScNDT. Localization studies with green fluorescent protein fusion proteins provided evidence that AtNDT1 resides in chloroplasts, whereas only AtNDT2 locates to mitochondria. Heterologous expression in Escherichia coli followed by purification, reconstitution in proteoliposomes, and uptake experiments revealed that both carriers exhibit a submillimolar affinity for NAD ؉ and transport this compound in a counterexchange mode. Among various substrates ADP and AMP are the most efficient counter-exchange substrates for NAD ؉ .Atndt1-and Atndt2-promoter-GUS plants demonstrate that both genes are strongly expressed in developing tissues and in particular in highly metabolically active cells. The presence of both carriers is discussed with respect to the subcellular localization of de novo NAD ؉ biosynthesis in plants and with respect to both the NAD ؉ -dependent metabolic pathways and the redox balance of chloroplasts and mitochondria.Nucleotides are metabolites of enormous importance for all living cells. They are the essential building blocks for DNA and RNA synthesis, energize most anabolic and many catabolic reactions, and fulfill critical functions in intracellular signal transduction (1, 2). Moreover, nucleotides serve as cofactors for a wide number of enzymes and are, with water, the most highly connected compounds within the metabolic network (3). Among these co-factors nicotinamide adenine dinucleotides are widely used for reductive/oxidative processes, playing important roles in the operation and control of a wide range of dehydrogenase activities. Accordingly, nucleotides are essential in nearly all cell organelles, and transport of these solutes into mitochondria, plastids, the endoplasmic reticulum, the Golgi apparatus, and peroxisomes has been observed (4 -7).Two types of nucleotide transport proteins have been identified to date at the molecular level: nucleotide transporter (NTT) 2 type carriers and members of the mitochondrial carrier family. The former transporters occur in plastids from all plants (8) and in a limited number of intracellular pathogenic bacteria (9). Most NTT-type carrier proteins catalyze an ATP/ADPϩP i counter-exchange mode of transport (10 -13), but several bacterial NTT proteins mediate either H ϩ /nucleotide transport or NAD ϩ /ADP counter-exchange (12,14,15). With the exception of the bacterial NAD ϩ /ADP carrier (14), all NTT proteins exhibit 12 predicted trans-membrane domains, whereas none of the NTT proteins share structural or domain similarities to members of the mitochondrial carrier family (11).Carriers belonging to the mitochondrial carrier family (MC...

“…The first mechanism resembles the "stop-transfer" pathways that are described for co-translational insertion of membrane proteins at the endoplasmic reticulum and posttranslational insertion of most nucleus-encoded mitochondria inner membrane proteins (15)(16)(17). In this model, the IEM protein is laterally released by the TIC translocon concurrently with its import across the envelope (18).…”

mentioning

confidence: 99%

Determinants for Stop-transfer and Post-import Pathways for Protein Targeting to the Chloroplast Inner Envelope Membrane

Viana

Schnell

2010

The inner envelope membrane (IEM) of the chloroplast plays key roles in controlling metabolite transport between the organelle and cytoplasm and is a major site of lipid and membrane synthesis within the organelle. IEM biogenesis requires the import and integration of nucleus-encoded membrane proteins. Previous reports have led to the conclusion that membrane proteins are inserted into the IEM during protein import from the cytoplasm via a stop-transfer mechanism or are completely imported into the stroma and then inserted into the IEM in a post-import mechanism. In this study, we examined the determinants for each pathway by comparing the targeting of APG1 (albino or pale green mutant 1), an example of a stoptransfer substrate, and atTic40, an example of a post-import substrate. We show that the APG1 transmembrane domain is sufficient to direct stop-transfer insertion. The APG1 transmembrane domain also functions as a topology determinant. We also show that the ability of the post-import signals within atTic40 to target proteins to the IEM is dependent upon their context within the full protein sequence. In the incorrect context, the atTic40 signals can behave as stop-transfer signals or fail to target fusion proteins to the IEM. These data suggest that the post-import pathway signals are complex and have evolved to avoid stop-transfer insertion.The chloroplast is a structurally complex organelle that performs diverse metabolic functions (1). It is composed of six distinguishable compartments, including three membranes (the outer envelope membrane, the inner envelope membrane, and the thylakoid membrane) and three aqueous and hydrophilic compartments (the intermembrane space (IMS), 2 located between the two envelope membranes, the stroma, and the thylakoid lumen) (2). These compartments are dependent upon the import and proper suborganellar targeting of several thousand nucleus-encoded proteins (3).The translocon of the outer envelope membrane of chloroplasts (TOC) and translocon of the inner envelope membrane of chloroplasts (TIC) complexes interact to mediate the import of the vast majority of nucleus-encoded proteins from the cytoplasm into the chloroplast stroma (4, 5). The TOC-TIC system recognizes the intrinsic N-terminal transit peptides of newly synthesized preproteins and catalyzes translocation across both envelope membranes simultaneously (2, 6, 7). In the case of thylakoid-targeted proteins, the targeting signals are bipartite. In addition to a transit peptide, thylakoid proteins contain secondary signals that target them from the stroma to the thylakoid. These processes occur in two independent steps (8, 9). The targeting of proteins to the thylakoid resembles protein export in bacteria because they utilize similar mechanisms (10, 11). For that reason, these pathways are referred to as "conservative sorting."Much less is known about the mechanisms of protein targeting and insertion at the chloroplast envelope membranes. This is surprising, considering the central role of the envelope in cellular me...