Kenneth Keegstra

Envelope membrane proteins that interact with chloroplastic precursor proteins.

The post-translational transport of cytoplasmically synthesized precursor proteins into chloroplasts requires proteins in the envelope membranes. To identify some of these proteins, label transfer cross-linking was performed using precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase (prSSU) that was blocked at an early stage of the transport process. Two envelope proteins were identified: an 86-kD protein and a 75-kD protein, both present in the outer membrane. Labeling of both proteins required prSSU and could not be accomplished with SSU lacking a transit peptide. Labeling of the 75-kD protein occurred only when low levels of ATP were present, whereas labeling of the 86-kD protein occurred in the absence of exogenous ATP. Although both labeled proteins were identified as proteins of the outer envelope membrane, the labeled form of the 75-kD protein could only be detected in fractions containing mixed envelope membranes. Based on these observations, we propose that prSSU first binds in an ATP-independent fashion to the 86-kD protein. The energy-requiring step is association with the 75-kD protein and assembly of a translocation contact site between the inner and outer membrane of the chloroplastic envelope.

The role of the transit peptide in the routing of precursors toward different chloroplast compartments

The role of the transit peptide in the routing of imported proteins inside the chloroplast was investigated with chimeric proteins in which the transit peptides for the nuclear-encoded ferredoxin and plastocyanin precursors were exchanged. Import and localization experiments with a reconstituted chloroplast system show that the ferredoxin transit peptide directs mature plastocyanin away from its correct location, the thylakoid lumen, to the stroma. With the plastocyanin transit peptide-mature ferredoxin chimera, a processing intermediate is arrested on its way to the lumen. We propose a two domain hypothesis for the plastocyanin transit peptide: the first domain functions in the chloroplast import process, whereas the second is responsible for transport across the thylakoid membrane. Thus, the transit peptide not only targets proteins to the chloroplast, but also is a major determinant in their subsequent localization within the organelle.

Characterization of a cDNA clone encoding a chloroplast-targeted Clp homologue

Effords to identify cDNA clones encoding chloroplastic envelope membrane proteins of Pisum sativum L. led to the isolation of a clone encoding a 92 kDa protein found in both the inner envelope membrane and the soluble fraction of chloroplasts. Sequential transcription and translation from the insert of this clone yielded a 102 kDa protein that could be imported into chloroplasts and processed to a 92 kDa form. Although the protein was identified because it reacted with antibodies to chloroplastic envelope proteins, the imported 92 kDa protein was recovered primarily in the soluble fraction of chloroplasts. The deduced amino acid sequence of this protein has strong similarity to the Clp proteins, a recently described family of highly conserved proteins present in all organisms examined to date. The physiological significance of the presence of this protein in chloroplasts is discussed.

Molecular cloning of a chloroplastic proteinassociated with both the envelope and thylakoid membranes

Chloroplasts consist of six morphologically distinct compartments. Each compartment has a specific set of polypeptides that perform distinct biochemical functions. We report here the identification of a membrane-associated protein with a novel localization. This protein was synthesized as a 37 kDa precursor and was processed to a mature protein of 30 kDa after being imported into isolated pea chloroplasts. Fractionation of chloroplasts showed that the 30 kDa mature protein was associated with both of the envelope membranes as well as with thylakoid membranes. Immunocyto-chemical localization of the 30 kDa protein revealed that the protein occurred in clusters in the vicinity of both the envelope and the thylakoid. Possible functions of this 30 kDa protein, inferred from its novel localization pattern, are discussed.

Lipid-peptide interactions between fragments of the transit peptide of ribulose-1,5-bisphosphate carboxylase/oxygenase and chloroplast membrane lipids.

The interactions of fragments of the transit peptide of ribulose‐1,5‐bisphosphate carboxylase/oxygenase with lipid monolayers was studied in order to investigate the possible involvement of the membrane lipids in the protein import process. The fragments are surface active and have a differential ability to insert in lipid monolayers. The fragments have a preference for the chloroplast galacto‐ and sulpholipids and phosphatidylglycerol and interact with envelope membrane lipid extracts. These results suggest that probably transit peptide—lipid interactions are involved in the chloroplast protein import process.

In vitro reconstitution of protein transport into chloroplasts.

Publisher Summary This chapter discusses various steps required for the in vitro reconstitution of protein transport into chloroplasts, the first being the production of precursor proteins. This is normally accomplished with an in vitro translation system, but other methods are also possible. The second step is isolation and purification of intact chloroplasts. Then, the precursor proteins and chloroplasts are incubated together under conditions that will allow transport to occur. The transport reaction is terminated by separating the chloroplasts, containing imported proteins, from the reaction mixture that contains residual precursors. It is possible to fractionate the recovered chloroplasts to determine which chloroplastic compartment contains the imported protein. Finally, the extent of transport is determined by measuring the amount of mature protein accumulated inside the chloroplasts. Transport has been reconstituted with many different precursors and with chloroplasts from several different species. The procedures that are used to study the transport of precursors into pea chloroplasts are discussed in the chapter. The products are then analyzed by electrophoretic separation of products and quantitative analysis of import products.

Transport of Proteins into Chloroplasts

The import of cytoplasmically synthesized proteins into chloroplasts involves an interaction between at least two components; the precursor protein, and the import apparatus in the chloroplast envelope membrane. This review summarizes the information available about each of these components. Precursor proteins consist of an amino terminal transit peptide attached to a passenger protein. Transit peptides from various precurosrs are diverse with respect to length and amino acid sequence; analysis of their sequences has not revealed insight into their mode of action. A variety of foreign passenger proteins can be imported into chloroplasts when a transit peptide is present at the amino terminus. However, foreign passenger proteins are not imported as efficiently as natural passenger proteins, and some chimeric precursor proteins are not imported into chloroplasts at all. Therefore, the passenger protein, as well as the transit peptide, influences the import process. Import begins by binding of the precursor to the chloroplast surface. It has been suggested that this binding is mediated by a receptor, but evidence to support this hypothesis remains incomplete and a receptor protein has not yet been characterized. Protein translocation requires energy derived from ATP hydrolysis, although there are conflicting reports as to where hydrolysis occurs and it is unclear how this energy is utilized. The mechanism(s) whereby proteins are translocated across either the two envelope membranes or the thylakoid membrane is not known.

Localization of Wheat Germ Agglutinin—Like Lectins in Various Species of the Gramineae

Antigenically similar chitin-binding lectins are present in the embryos of wheat, barley, and rye, members of the Triticeae tribe of the grass family (Gramineae). However, the lectins display different localization patterns in these embryos. Lectin is absent from the coleoptile of barley but is present in the outer surface cells of this organ in wheat and in both inner and outer surface cells of rye coleoptiles. All three cereals contain lectin at the periphery of embryonic roots. Similar lectins were not detected in oats and pearl millet, members of other tribes of the Gramineae. Rice, a species only distantty related to wheat, contains a lectin that is antigenically similar to the other cereal lectins and located at the periphery of embryonic roots and throughut the coleoptile.

Current views on chloroplast protein import and hypotheses on the origin of the transport mechanism

Most chloroplastic proteins are synthesized as precursors in the cytosol prior to their transport into chloroplasts. These precursors are generally synthesized in a form that is larger than the mature form found inside chloroplasts. The extra amino acids, called transit peptides, are present at the amino terminus. The transit peptide is necessary and sufficient to recognize the chloroplast and induce movement of the attached protein across the envelope membranes. In this review, we discuss the primary and secondary structure of transit peptides, describe what is known about the import process, and present some hypotheses on the evolutionary origin of the import mechanism.

Chloroplast import characteristics of chimeric proteins

We have examined the import of a series of chimeric precursor proteins into chloroplasts. These fusion proteins contained the transit peptide, and various amounts of the amino-terminal region of the mature peptide, from the small subunit of ribulose 1,5-bisphosphate carboxylase, linked to the coat protein of brome mosaic virus. Chimeric genes were cloned into SP6 plasmids and in vitro transcription/translation was used to produce fusion proteins, which were examined in a quantitative in vitro import assay. A chimeric protein which contained only the transit peptide fused to the coat protein was imported into chloroplasts. A second chimeric precursor, which also contained a small portion of the mature peptide, was imported at nearly the same rate. A chimeric protein which contained the transit peptide and most of the mature peptide fused to the coat protein was not imported. These results suggest that secondary or tertiary structural features of precursor proteins are important for protein import, and that the presence of a transit peptide in a protein does not necessarily ensure import of that protein into chloroplasts.