Biomedical subjects

K Keegstra

Publications and source records attributed to K Keegstra.

At least 37 records · Page 2Linked to original sources

PubMed1997

Stable association of chloroplastic precursors with protein translocation complexes that contain proteins from both envelope membranes and a stromal Hsp100 molecular chaperone.

Cytoplasmically synthesized precursors interact with translocation components in both the outer and inner envelope membranes during transport into chloroplasts. Using co-immunoprecipitation techniques, with antibodies specific to known translocation components, we identified stable interactions between precursor proteins and their associated membrane translocation components in detergent-solubilized chloroplastic membrane fractions. Antibodies specific to the outer envelope translocation components OEP75 and OEP34, the inner envelope translocation component IEP110 and the stromal Hsp100, ClpC, specifically co-immunoprecipitated precursor proteins under limiting ATP conditions, a stage we have called docking. A portion of these same translocation components was co-immunoprecipitated as a complex, and could also be detected by co-sedimentation through a sucrose density gradient. ClpC was observed only in complexes with those precursors utilizing the general import apparatus, and its interaction with precursor-containing translocation complexes was destabilized by ATP. Finally, ClpC was co-immunoprecipitated with a portion of the translocation components of both outer and inner envelope membranes, even in the absence of added precursors. We discuss possible roles for stromal Hsp100 in protein import and mechanisms of precursor binding in chloroplasts.

Adenosine Triphosphate↗

PubMed1996

Topology of IEP110, a component of the chloroplastic protein import machinery present in the inner envelope membrane.

Proteins from both the inner and outer envelope membranes are engaged in the recognition and translocation of precursor proteins into chloroplasts. A 110 kDa protein of the chloroplastic inner envelope membrane was identified as a component of the protein import apparatus by two methods. First, this protein was part of a 600 kDa complex generated by cross-linking of precursors trapped in the translocation process. Second, solubilization with detergents of chloroplasts containing trapped precursors resulted in the identification of a complex containing both radiolabeled precursor and IEP110. Trypsin treatment of intact purified chloroplasts was used to study the topology of IEP110. The protease treatment left the inner membrane intact while simultaneously degrading domains of inner envelope proteins exposed to the intermembrane space. About 90 kDa of IEP110 was proteolitically removed, indicating that large portions protrude into the intermembrane space. Hydropathy analysis of the protein sequence deduced from the isolated cDNA clone in addition to Western blot analysis using an antiserum of IEP110 specific to the N-terminal 20 kDa, suggests that the N-terminus serves to anchor the protein in the membrane. We speculate that IEP110 could be involved in the formation of translocation contact sites due to its specific topology.

Amino Acid Sequence↗

PubMed1996

A novel, bipartite transit peptide targets OEP75 to the outer membrane of the chloroplastic envelope.

OEP75 is an outer envelope membrane component of the chloroplastic protein import apparatus and is synthesized in the cytoplasm as a higher molecular weight precursor (prOEP75). During its own import, prOEP75 is processed first to an intermediate (iOEP75) and subsequently to the mature form (mOEP75). Experiments conducted with stromal extracts indicated that iOEP75 was generated from prOEP75 by the activity of the stromal processing peptidase. The specific processing site was determined and used to divide the prOEP75 transit peptide into N- and C-terminal domains. To determine the targeting functions of the two domains of the transit peptide and of the mature region of prOEP75, we created a deletion mutant construct from prOEP75 and chimeric constructs between domains of prOEP75 and the precursor to a small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Analysis of these constructs by in vitro chloroplastic protein import assays revealed that the transit peptide of prOEP75 is bipartite in that the N- and C-terminal portions contain chloroplastic and intraorganellar targeting information, respectively.

Amino Acid Sequence↗

PubMed1995

A component of the chloroplastic protein import apparatus is targeted to the outer envelope membrane via a novel pathway.

A chloroplastic outer envelope membrane protein of 75 kDa (OEP75) was identified previously as a component of the protein import machinery. Here we provide additional evidence that OEP75 is a component of protein import, present the isolation of a cDNA clone encoding this protein, briefly describe its developmental expression and tissue specificity, and characterize its insertion into the outer envelope membrane. OEP75 was synthesized as a higher molecular weight precursor (prOEP75) which bound to isolated chloroplasts in an in vitro import assay and subsequently was processed to the mature form (mOEP75). During this import assay, two proteins intermediate in size between prOEP75 and mOEP75 were detected. One of these intermediates was also detected in chloroplast envelopes isolated from young pea leaves. Binding and processing of prOEP75 required ATP and one or more surface-exposed proteinaceous components, and was competed by prSSU, a stromal-targeted protein. We propose that the N-terminus of the prOEP75 transit peptide acts as a stromal-targeting domain and a central, hydrophobic region of this transit peptide acts as a stop-transfer domain. A complex route of insertion and processing of prOEP75 may exist to ensure high fidelity targeting of this import component.

Adenosine Triphosphate↗

PubMed1994

Methotrexate does not block import of a DHFR fusion protein into chloroplasts.

Protein import into chloroplasts requires the movement of a precursor protein across the envelope membranes. The conformation of a precursor as it passes from the aqueous medium across the hydrophobic membranes is not known in detail. To address this problem we examined precursor conformation during translocation using the chimeric precursor PCDHFR, which contains the plastocyanin (PC) transit peptide in front of mouse cytosolic dihydrofolate reductase (DHFR). The chimeric protein is targeted to chloroplasts and is competent for import. The conformation of PCDHFR can be stabilized by complexing with methotrexate, an analogue of the substrate of DHFR. Methotrexate strongly inhibits DHFR import into yeast mitochondria (M. Eilers and G. Schatz, Nature 322 (1986) 228-232), presumably because the precursor must unfold to cross the membrane and it cannot do so when complexed with methotrexate. We show here that methotrexate does not block PCDHFR import into chloroplasts. Methotrexate does slow the rate of import, and protects DHFR from degradation once inside chloroplasts. The processed protein is localized in the stroma, indicating that import into thylakoids is impeded. Protease sensitivity assays indicate that the complex of precursor protein with methotrexate changes in conformation during the translocation across the envelope.

Amino Acid Sequence↗

PubMed1994

Molecular cloning of a chloroplastic protein associated 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.

Amino Acid Sequence↗

PubMed1994

Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones.

High-throughput automated partial sequencing of anonymous cDNA clones provides a method to survey the repertoire of expressed genes from an organism. Comparison of the coding capacity of these expressed sequence tags (ESTs) with the sequences in the public data bases results in assignment of putative function to a significant proportion of the ESTs. Thus, the more than 13,400 plant ESTs that are currently available provide a new resource that will facilitate progress in many areas of plant biology. These opportunities are illustrated by a description of the results obtained from analysis of 1500 Arabidopsis ESTs from a cDNA library prepared from equal portions of poly(A+) mRNA from etiolated seedlings, roots, leaves, and flowering inflorescences. More than 900 different sequences were represented, 32% of which showed significant nucleotide or deduced amino acid sequences similarity to previously characterized genes or proteins from a wide range of organisms. At least 165 of the clones had significant deduced amino acid sequence homology to proteins or gene products that have not been previously characterized from higher plants. A summary of methods for accessing the information and materials generated by the Arabidopsis cDNA sequencing project is provided.

Amino Acid Sequence↗

PubMed1994

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.

Adenosine Triphosphate↗

PubMed1993

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

Efforts 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.

Amino Acid Sequence↗

PubMed1993

Analysis of chloroplast transit peptide function using mutations in the carboxyl-terminal region.

Protein import into chloroplasts requires a transit peptide, which interacts with the chloroplast transport apparatus and leads to translocation of the protein across the chloroplast envelope. While the amino acid sequences of many transit peptides are known, functional domains have been difficult to identify. Previous studies suggest that the carboxyl terminus of the transit peptide for ribulose bisphosphate carboxylase small subunit is important for both translocation across the chloroplast envelope and proper processing of the precursor protein. We dissected this region using in vitro mutagenesis, creating a set of mutants with small changes in primary structure predicted to cause alterations in secondary structure. The import behavior of the mutant proteins was assessed using isolated chloroplasts. Our results show that removal of a conserved arginine residue in this region results in impaired processing, but does not necessarily affect import rates. In contrast, substituting amino acids with low reverse turn or amphiphilic potential for other original residues affected import rate but not processing.

Amino Acid Sequence↗

PubMed1992

Information for targeting to the chloroplastic inner envelope membrane is contained in the mature region of the maize Bt1-encoded protein.

Based on the protein sequence deduced from a cDNA clone, it has been proposed that the maize bt1 locus encodes an amyloplast membrane metabolite translocator protein (Sullivan, T. D., Strelow, L. I., Illingworth, C. A., Phillips, R. L., and Nelson, O. E., Jr. (1991) Plant Cell 3, 1337-1348). The present work provides further evidence for this hypothesis by showing that the gene product of Bt1 could be imported into chloroplasts in vitro and processed to lower molecular weight mature proteins. More importantly, the imported mature proteins were localized to the inner envelope membrane, where metabolite translocators are located in plastids. In addition, the location of information for targeting to the inner membrane was investigated by constructing and analyzing the import of chimeric precursor proteins. A chimeric protein with the transit peptide of the precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase fused to the mature region of the Bt1-encoded protein was targeted to the inner envelope membrane of chloroplasts. Moreover, a chimeric protein with the transit peptide of the Bt1-encoded protein fused to the mature protein of the light-harvesting chlorophyll a/b binding protein was targeted to the thylakoid. These results indicate that the transit peptide of the Bt1-encoded protein functions primarily as a stromal targeting sequence. The information for targeting to the chloroplastic inner envelope membrane is contained in the mature region of the protein.

Base Sequence↗

PubMed1992

Isolation of a carotenoid-containing sub-membrane particle from the chloroplastic envelope outer membrane of pea (Pisum sativum).

Chloroplastic envelope membranes isolated from pea (Pisum sativum) leaves are rich in carotenoids, containing approximately 2 micrograms of carotenoid mg-1 protein. We report here that envelopes can be surfactant-solubilized while maintaining association of carotenoids with protein components of the membrane. Treatment of isolated chloroplastic envelope membranes with 0.5% Deriphat 160 (N-lauryl-beta-imminodipropionate) causes general solubilization but preserves an envelope sub-membrane fragment which is fractionated by centrifugation in a sucrose gradient and by chromatography on a column of DEAE-Sephacel. The isolated submembrane complex contained five major proteins with M(r) values equivalent to 75,000, 36,000, 34,000 17,500, and 14,500. Spectroscopic and chromatographic analyses revealed that the complex contains violaxanthin and at least one other carotenoid. Carotenoid content of the fractionated complex was estimated as 4.8 micrograms mg-1 protein. Immunoblot analysis reveals that the constituent proteins of this complex are derived from the chloroplastic outer envelope membrane. These data suggest that at least some of the carotenoids of the chloroplastic envelope may be organized by apoproteins.

Carotenoids↗

PubMed1992

The binding of precursor proteins to chloroplasts requires nucleoside triphosphates in the intermembrane space.

Protein import into chloroplasts is initiated by a binding interaction between a precursor protein and the surface of the outer envelope. The binding step was previously shown to be energy-dependent (Olsen, L. J., Theg, S. M., Selman, B. R., and Keegstra, K. (1989) J. Biol. Chem. 264, 6724-6729). We took advantage of the broad nucleotide specificity of the energy requirement for binding to investigate the site of the nucleoside triphosphate (NTP) requirement. GTP supported precursor binding to chloroplasts. It was not converted to ATP, as determined by direct ATP measurements, and was not transported across the inner envelope. Thus, GTP supported binding from either the intermembrane space or outside the outer membrane. To distinguish between an intermembrane space and an external NTP requirement, we experimentally manipulated the NTP levels inside and outside chloroplasts. Internally generated ATP was able to support binding in the presence of an external membrane-impermeant ATP trap. Therefore, since GTP supported binding from either the intermembrane space or outside the chloroplast, and ATP supported binding from either the intermembrane space or the stroma, we concluded that the site of NTP utilization for precursor binding to chloroplasts was the intermembrane space between the two envelope membranes.

Adenosine Triphosphate↗

PubMed1992

Isolation and characterization of a cDNA clone encoding the major hsp70 of the pea chloroplastic stroma.

The 70-kD heat shock proteins (hsp70s) are a group of ubiquitous, highly conserved molecular chaperones that have been implicated in a variety of processes, ranging from DNA replication to protein folding and transport. To learn more about the evolution and possible functions of higher plant chloroplastic hsp70s, we isolated a cDNA clone encoding the major stromal hsp70 of pea chloroplasts, which we term CSS1 (Chloroplastic Stress Seventy). This cDNA clone encodes a 75,490-D protein that is very closely related to an hsp70 from the cyanobacterium, Synechocystis. CSS1 is nuclear encoded and synthesized as a higher molecular mass precursor with a chloroplastic transit peptide approximately 65 amino acids long. CSS1 mRNA was detected in RNA samples from leaves and roots of pea (Pisum sativum) plants grown at 18 degrees C but increased 9- and 6-fold, respectively, after brief exposure of the plants to elevated temperature. We discuss the possible role(s) of CSS1 in chloroplastic protein transport and other processes.

Journal Article↗

PubMed1991

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.

Amino Acid Sequence↗

PubMed1991

Synthetic analogues of a transit peptide inhibit binding or translocation of chloroplastic precursor proteins.

Although amino-terminal transit peptides of chloroplastic precursor proteins are known to be necessary and sufficient for import into chloroplasts, the mechanism by which they mediate this process is not understood. Another important question is whether different precursors share a common transport apparatus. We used 20-residue synthetic peptides corresponding to regions of the transit peptide of the precursor to the small subunit of ribulose bisphosphate carboxylase (prSS) as competitive inhibitors for the binding and translocation of precursor proteins into chloroplasts. Synthetic peptides with sequences corresponding to either end of the transit peptide had little to no effect on binding of prSS to chloroplasts, but significantly inhibited its translocation. Synthetic peptides corresponding to the central region of the transit peptide inhibited binding of prSS to chloroplasts. Each of the peptides inhibited binding or translocation of precursors to light-harvesting chlorophyll a/b protein, ferredoxin, and plastocyanin in the same manner and to a similar extent as prSS transport was inhibited. The results presented in this paper suggest that the central regions of the transit peptide of prSS mediate binding to the chloroplastic surface, whereas the ends of this transit peptide are more important for translocation across the envelope. Furthermore, all of the precursors tested appear to share components in the transport apparatus even though they are sorted to different chloroplastic compartments.

Amino Acid Sequence↗

PubMed1991

Full-length plastocyanin precursor is translocated across isolated thylakoid membranes.

In higher plants, the chloroplastic protein plastocyanin is synthesized as a transit peptide-containing precursor by cytosolic ribosomes and posttranslationally transported to the thylakoid lumen. En route to the lumen, a plastocyanin precursor is first imported into chloroplasts and then further directed across the thylakoid membrane by a second distinct transport event. A partially processed form of plastocyanin is observed in the stroma during import experiments using intact chloroplasts and has been proposed to be the translocation substrate for the second step (Smeekens, S., Bauerle, C., Hageman, J., Keegstra, K., and Weisbeek, P. (1986) Cell 46, 365-375). To further characterize this second step, we have reconstituted thylakoid transport in a system containing in vitro-synthesized precursor proteins and isolated thylakoid membranes. This system was specific for lumenal proteins since stromal proteins lacking the appropriate targeting information did not accumulate in the thylakoid lumen. Plastocyanin precursor was taken up by isolated thylakoids, proteolytically processed to mature size, and converted to holo form. Translocation was temperature-dependent and was stimulated by millimolar levels of ATP but did not strictly require the addition of stromal factors. We have examined the substrate requirements of thylakoid translocation by testing the ability of different processed forms of plastocyanin to transport in the in vitro system. Interestingly, only the full-length plastocyanin precursor, not the partially processed intermediate form, was competent for transport in this in vitro system.

Adenosine Triphosphate↗

PubMed1991

Kinetic analysis of the transport of thylakoid lumenal proteins in experiments using intact chloroplasts.

The transport of the lumenal protein plastocyanin has been proposed to occur in two steps: 1) transport across the chloroplastic envelope to the stroma and 2) transport across the thylakoid membrane to the lumen where proteolytic maturation occurs. A partially processed stromal form of plastocyanin has been tentatively identified as a pathway intermediate and as the substrate for the second translocation step (Smeekens, S., Bauerle, C., Hageman, J., Keegstra, K., and Weisbeek, P. (1986) Cell 46, 365-375). In this study, we have examined the transport kinetics of several lumenal proteins under various incubation conditions. Soluble intermediate sized forms were observed in import reactions with plastocyanin precursors from three higher plant species and with the precursor of the 33-kDa polypeptide of the oxygen-evolving enhancer complex. The accumulation patterns observed for these soluble intermediate sized forms depended on incubation conditions and could not be consistently explained by a simple model where the intermediate sized form is the substrate for the second step. Thus, it has not been possible to clearly identify the substrate for thylakoid translocation in organello.

Amino Acid Sequence↗