Targeting of glyoxysomal proteins to peroxisomes in leaves and roots of a higher plant.

Higher plants possess several classes of peroxisomes that are present at distinct developmental stages and serve different metabolic roles. To investigate the cellular processes that regulate developmental transitions of peroxisomal function, we analyzed the targeting of glyoxysomal proteins to leaf-type and root peroxisomes. We transferred genes encoding the glyoxysome-specific enzymes isocitrate lyase (IL) and malate synthase into Arabidopsis plants and showed, in cell fractionation and immunogold localization experiments, that the glyoxysomal proteins were imported into leaf-type and root peroxisomes. We next defined the sequences that target IL to peroxisomes and asked whether the same targeting determinant is recognized by different classes of the organelle. By localizing deletion and fusion derivatives of IL, we showed that the polypeptide's carboxyl terminus is both necessary for its transport to peroxisomes and sufficient to redirect a passenger protein from the cytosol to both glyoxysomes and leaf-type peroxisomes. Thus, glyoxysomal proteins are transported into several classes of peroxisomes using a common targeting determinant, suggesting that protein import does not play a regulatory role in determining a peroxisome's function. Rather, the specific metabolic role of a peroxisome appears to be determined primarily by processes that regulate the synthesis and/or stability of its constituent proteins. These processes are specified by the differentiated state of the cells in which the organelles are found.

Protein-specific energy requirements for protein transport across or into thylakoid membranes. Two lumenal proteins are transported in the absence of ATP.

Cytosolically synthesized thylakoid proteins must be translocated across the chloroplast envelope membranes, traverse the stroma, and then be translocated into or across the thylakoid membrane. Protein transport across the envelope requires ATP hydrolysis but not electrical or proton gradients. The energy requirements for the thylakoid translocation step were studied here for the light-harvesting chlorophyll a/b protein (LHCP), an integral membrane protein, and for several thylakoid lumen-resident proteins: plastocyanin and OE33, OE23, and OE17 (the 33-, 23-, and 17-kDa subunits of the oxygen-evolving complex, respectively). Dissipation of the thylakoid protonmotive force during an in organello protein import assay partially inhibited the thylakoid localization of LHCP and OE33, totally inhibited localization of OE23 and OE17, and had no effect on localization of plastocyanin. We used reconstitution assays for LHCP insertion and for OE23 and OE17 transport into isolated thylakoids to investigate the energy requirements in detail. The results indicated that LHCP insertion absolutely requires ATP hydrolysis and is enhanced by a transthylakoid delta pH and that transport of OE23 and OE17 is absolutely dependent upon a delta pH. Surprisingly, OE23 and OE17 transport occurred maximally in the complete absence of ATP. These results establish the thylakoid membrane as the only membrane system in which a delta pH can provide all of the energy required to translocate proteins across the bilayer. They also demonstrate that the energy requirements for integration into or translocation across the thylakoid membranes are protein-specific.

Physiologically active chloroplasts contain pools of unassembled extrinsic proteins of the photosynthetic oxygen-evolving enzyme complex in the thylakoid lumen.

The oxygen-evolving complex (OEC) of photosystem II (PS II) consists of at least three extrinsic membrane-associated protein subunits, OE33, OE23, and OE17, with associated Mn2+, Ca2+, and Cl- ions. These subunits are bound to the lumen side of PS II core proteins embedded in the thylakoid membrane. Our experiments reveal that a significant fraction of each subunit is normally present in unassembled pools within the thylakoid lumen. This conclusion was supported by immunological detection of free subunits after freshly isolated pea thylakoids were fractionated with low levels of Triton X-100. Plastocyanin, a soluble lumen protein, was completely released from the lumen by 0.04% Triton X-100. This gentle detergent treatment also caused the release from the thylakoids of between 10 and 20%, 40 and 60%, and 15 and 50% of OE33, OE23, and OE17, respectively. Measurements of the rates of oxygen evolution from Triton-treated thylakoids, both in the presence and absence of Ca2+, and before and after incubation with hydroquinone, demonstrated that the OEC was not dissociated by the detergent treatment. Thylakoids isolated from spinach released similar amounts of extrinsic proteins after Triton treatment. These data demonstrate that physiologically active chloroplasts contain significant pools of unassembled extrinsic OEC polypeptide subunits free in the lumen of the thylakoids.