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

Abstract

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...

Targeting of a polytopic membrane protein to the inner envelope membrane of chloroplasts in vivo involves multiple transmembrane segments

“…Time-course import assays were performed using intact pea chloroplasts as described previously (Viana et al , 2010). The quantitative analysis of radiolabeled samples was performed with a Typhoon FLA7000 PhosphorImager and ImageQuant TL software (GE Healthcare Life Sciences).…”

Common and Specific Protein Accumulation Patterns in Different Albino/Pale-Green Mutants Reveals Regulon Organization at the Proteome Level

“…However, the specific absence of two subsequent enzymes of the oxidative pentose phosphate pathway, Glc-6-P dehydrogenase (AT5G35790) and 6-phosphogluconolactonase3 (AT5G24400), suggests differences in the heterotrophic metabolism in apg1 compared with the other apg mutants. Interestingly, Dit1 is transcriptionally coregulated with Tic21 (ATTED-II), suggesting a functional connection between these proteins that is currently not understood (Obayashi et al, 2009;Viana et al, 2010). Further surprising is the accumulation of PSAH in apg1 that reaches beyond wild-type levels, despite the otherwise decreased accumulation of thylakoid membrane proteins in apg1.…”

Map-based cloning of a recessive genic male sterility locus in Brassica napus L. and development of its functional marker

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