Biomedical subjects

K Cline

Publications and source records attributed to K Cline.

At least 37 records · Page 2Linked to original sources

PubMed1996

Import and routing of nucleus-encoded chloroplast proteins.

Most chloroplast proteins are nuclear encoded, synthesized as larger precursor proteins in the cytosol, posttranslationally imported into the organelle, and routed to one of six different compartments. Import across the outer and inner envelope membranes into the stroma is the major means for entry of proteins destined for the stroma, the thylakoid membrane, and the thylakoid lumen. Recent investigations have identified several unique protein components of the envelope translocation machinery. These include two GTP-binding proteins that appear to participate in the early events of import and probably regulate precursor recognition and advancement into the translocon. Localization of imported precursor proteins to the thylakoid membrane and thylakoid lumen is accomplished by four distinct mechanisms; two are homologous to bacterial and endoplasmic reticulum protein transport systems, one appears unique, and the last may be a spontaneous mechanism. Thus chloroplast protein targeting is a unique and surprisingly complex process. The presence of GTP-binding proteins in the envelope translocation machinery indicates a different precursor recognition process than is present in mitochondria. Mechanisms for thylakoid protein localization are in part derived from the prokaryotic endosymbiont, but are more unusual and diverse than expected.

Amino Acid Sequence↗

PubMed1995

A chloroplast homologue of the signal recognition particle subunit SRP54 is involved in the posttranslational integration of a protein into thylakoid membranes.

The mechanisms involved in the integration of proteins into the thylakoid membrane are largely unknown. However, many of the steps of this process for the light-harvesting chlorophyll a/b protein (LHCP) have been described and reconstituted in vitro. LHCP is synthesized as a precursor in the cytosol and posttranslationally imported into chloroplasts. Upon translocation across the envelope membranes, the N-terminal transit peptide is cleaved, and the apoprotein is assembled into a soluble "transit complex" and then integrated into the thylakoid membrane via three transmembrane helices. Here we show that 54CP, a chloroplast homologue of the 54-kDa subunit of the mammalian signal recognition particle (SRP54), is essential for transit complex formation, is present in the complex, and is required for LHCP integration into the thylakoid membrane. Our data indicate that 54CP functions posttranslationally as a molecular chaperone and potentially pilots LHCP to the thylakoids. These results demonstrate that one of several pathways for protein routing to the thylakoids is homologous to the SRP pathway and point to a common evolutionary origin for the protein transport systems of the endoplasmic reticulum and the thylakoid membrane.

Animals↗

PubMed1995

Complete sequence of the citrus tristeza virus RNA genome.

The sequence of the entire genome of citrus tristeza virus (CTV), Florida isolate T36, was completed. The 19,296-nt CTV genome encodes 12 open reading frames (ORFs) potentially coding for at least 17 protein products. The 5'-proximal ORF 1a starts at nucleotide 108 and encodes a large polyprotein with calculated MW of 349 kDa containing domains characteristic of (from 5' to 3') two papain-like proteases (P-PRO), a methyltransferase (MT), and a helicase (HEL). Alignment of the putative P-PRO sequences of CTV with the related proteases of beet yellows closterovirus (BYV) and potyviruses allowed the prediction of catalytic cysteine and histidine residues as well as two cleavage sites, namely Val-Gly/Gly for the 5' proximal P-PRO domain and Met-Gly/Gly for the 5' distal P-PRO domain. The autoproteolytic cleavage of the polyprotein at these sites would release two N-terminal leader proteins of 54 and 55 kDa, respectively, and a 240-kDa C-terminal fragment containing MT and HEL domains. The apparent duplication of the leader domain distinguishes CTV from BYV and accounts for most of the size increase in the ORF 1a product of CTV. The downstream ORF 1b encodes a 57-kDa putative RNA-dependent RNA polymerase (RdRp), which is probably expressed via a +1 ribosomal frameshift. Sequence analysis of the frameshift region suggests that this +1 frameshift probably occurs at a rare arginine codon CGG and that elements of the RNA secondary structure are unlikely to be involved in this process. The complete polyprotein resulting from this frameshift event has a calculated MW of 401 kDa and after cleavage of the two N-terminal leaders would yield a 292-kDa protein containing the MT, HEL, and RdRp domains. Phylogenetic analysis of the three replication-associated domains, MT, HEL, and RdRp, indicates that CTV and BYV form a separate closterovirus lineage within the alpha-like supergroup of positive-strand RNA viruses. Two gene blocks or modules can be easily identified in the CTV genome. The first includes the replicative MT, HEL, and RdRp genes and is conserved throughout the entire alpha-like superfamily. The second block consists of five ORFs, 3 to 7, conserved among closteroviruses, including genes for the CTV homolog of HSP70 proteins and a duplicate of the coat protein gene. The 3'-terminal ORFs 8 to 11 encode a putative RNA-binding protein (ORF 11), and three proteins with unknown functions; this gene array is poorly conserved among closteroviruses.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1995

Are small firms greater health risks?

To test whether use of health care services is a function of firm size, we analyzed a three-year database (1988-1990) of private insurance claims, representing 28,990 firms and approximately 1.4 million subscribers in western Pennsylvania. In this database both small and large firms had higher medically underwritten costs than mid-size firms had. Furthermore, risk-pooling alternatives that included small companies had a lower cost per subscriber than the risk pools that included large companies, especially companies of more than 500 contract holders. Age, sex, health status, and the types of hospitals used for inpatient care of pooled subscribers, in combination, were found to be the important determinants of costs. With risk adjustment based on these factors to correct for adverse risk selection, community rating can be a feasible approach to increasing the affordability and accessibility of health insurance to the majority of those who lack it.

Adult↗

PubMed1994

SecA homolog in protein transport within chloroplasts: evidence for endosymbiont-derived sorting.

The SecA protein is an essential, azide-sensitive component of the bacterial protein translocation machinery. A SecA protein homolog (CPSecA) now identified in pea chloroplasts was purified to homogeneity. CPSecA supported protein transport into thylakoids, the chloroplast internal membrane network, in an azide-sensitive fashion. Only one of three pathways for protein transport into thylakoids uses the CPSecA mechanism. The use of a bacteria-homologous mechanism in intrachloroplast protein transport provides evidence for conservative sorting of proteins within chloroplasts.

Adenosine Triphosphatases↗

PubMed1994

Plastocyanin and the 33-kDa subunit of the oxygen-evolving complex are transported into thylakoids with similar requirements as predicted from pathway specificity.

Plastocyanin and the 33-kDa subunit of the oxygen-evolving complex (OE33) are two of several thylakoid lumen-located proteins that are made in the cytosol, imported into chloroplasts, and subsequently transported into thylakoids. Recently, competition studies showed that there are two pathways for protein transport into the thylakoid lumen and that plastocyanin and OE33 are on the same pathway (Cline, K., Henry, R., Li, C., and Yuan, J. (1993) EMBO J. 12, 4105-4114). Our expectation is that transport requirements reflect the steps of the process and that proteins on the same pathway share similar requirements. Unfortunately, the transport requirements for plastocyanin and OE33 are not well established. Here, we investigated transport in a reconstituted system with isolated thylakoids. Efficient transport of OE33 and plastocyanin was only obtained when stromal extract was included in the assay. Heat or protease treatment of stromal extract eliminated its ability to stimulate transport. Transport was abolished by treatments designed to deplete ATP or to prevent its formation and was greatly reduced in the presence of ionophores that dissipate the trans-thylakoidal proton gradient. These results show that transport of OE33 and plastocyanin requires ATP and is stimulated by stromal protein(s) and the trans-thylakoidal proton gradient. Taken together, these and previous results suggest that there are two mechanistically distinct pathways for protein transport into the thylakoid lumen.

Adenosine Triphosphate↗

PubMed1994

Differences between lumen targeting domains of chloroplast transit peptides determine pathway specificity for thylakoid transport.

Nuclear encoded thylakoid lumen proteins are imported into the chloroplast storma and further directed across thylakoid membranes by lumen targeting domains. Recently, we showed that there are two protein-specific pathways for transport into the lumen. This was unexpected in that lumen targeting domains have similar properties, all containing bacterial signal peptide motifs. Nevertheless, sequence homology analysis suggests that pathway specificity is determined by elements in the lumen targeting domain. To test this, we constructed and analyzed chimeric proteins in which transit peptides from proteins transported by one pathway were fused to the mature domains of proteins directed by the other. We also investigated the transport characteristics of a previously unexamined protein whose pathway was predicted by sequence similarity analysis. Our results confirm that lumen targeting domains contain pathway sorting elements and further indicate that distinct energy and stroma requirements for transport are pathway characteristics, unrelated to the passenger protein. These findings suggest the operation of two mechanistically different translocators.

Arabidopsis↗

PubMed1994

An Arabidopsis chloroplast RNA-binding protein gene encodes multiple mRNAs with different 5' ends.

An Arabidopsis cDNA (Atrbp33) encoding a nuclear-encoded chloroplast RNA-binding protein (RBP) has been isolated (A.J. DeLisle [1993] Plant Physiol 102: 313-314). ATRBP33 shares global structural homology with all known chloroplast RBPs: a chloroplast transit peptide in the amino terminus, followed by a unique acidic domain and a tandem pair of ribonucleoprotein consensus sequence-type RNA-binding domains in the carboxyl end. In vitro translation products of Atrbp33 were found to be imported into chloroplasts, suggesting that ATRBP33 is localized in chloroplasts. The expression of Atrbp33 was higher in chloroplast-containing organs than in nonchloroplast-containing organs. Furthermore, Atrbp33 was expressed in a light-dependent manner. These features are consistent with its postulated role in posttranscriptional control of chloroplast genes. Northern analyses and RNase protection assays showed that as many as nine messages are encoded by the single Atrbp33 gene. Sequence analysis of the cDNAs indicated that some of the transcripts have truncated 5' ends. Most interestingly, the multiple mRNAs potentially encode different polypeptides, one of which lacks a chloroplast transit peptide and acidic domain and contains only one intact RNA-binding domain. Unlike the chloroplast-localized ATRBP33, the truncated polypeptide may function in other cellular compartments.

Amino Acid Sequence↗

PubMed1993

Stromal factor plays an essential role in protein integration into thylakoids that cannot be replaced by unfolding or by heat shock protein Hsp70.

The light-harvesting chlorophyll a/b protein (LHCP) is an integral thylakoid membrane protein. It is made in the cytosol as a precursor (pLHCP), imported into chloroplasts, and subsequently integrated into thylakoids. Integration of pLHCP into thylakoids requires a stromal protein factor that functions in part to maintain the solubility and integration competence of pLHCP. Recently, it was reported that unfolded pLHCP was sufficient for integration and that the stromal factor, identified as the plastid Hsp70, was required only to prevent pLHCP refolding [Yalovsky, S., Paulsen, H., Michaeli, D., Chitnis, P. R. & Nechushtai, R. (1992) Proc. Natl. Acad. Sci. USA 89, 5616-5619]. Our studies, using more rigorous criteria for integration, show that unfolded pLHCP is not sufficient; stromal factor is an absolute requirement for integration. Furthermore, experiments with purified Hsp70 as well as Hsp70-depleted stromal extract demonstrate that Hsp70 is not the stromal factor. These results plus the finding that pLHCP diluted out of urea is relatively stable as a substrate for integration point to an additional role for the stromal factor in targeting and/or membrane translocation.

Antibodies↗

PubMed1993

Multiple pathways for protein transport into or across the thylakoid membrane.

Many thylakoid proteins are cytosolically synthesized and have to cross the two chloroplast envelope membranes as well as the thylakoid membrane en route to their functional locations. In order to investigate the localization pathways of these proteins, we over-expressed precursor proteins in Escherichia coli and used them in competition studies. Competition was conducted for import into the chloroplast and for transport into or across isolated thylakoids. We also developed a novel in organello method whereby competition for thylakoid transport occurred within intact chloroplasts. Import of all precursors into chloroplasts was similarly inhibited by saturating concentrations of the precursor to the OE23 protein. In contrast, competition for thylakoid transport revealed three distinct precursor specificity groups. Lumen-resident proteins OE23 and OE17 constitute one group, lumenal proteins plastocyanin and OE33 a second, and the membrane protein LHCP a third. The specificity determined by competition correlates with previously determined protein-specific energy requirements for thylakoid transport. Taken together, these results suggest that thylakoid precursor proteins are imported into chloroplasts on a common import apparatus, whereupon they enter one of several precursor-specific thylakoid transport pathways.

Base Sequence↗

PubMed1992

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.

Adenosine Triphosphate↗

PubMed1991

A stromal protein factor maintains the solubility and insertion competence of an imported thylakoid membrane protein.

The light-harvesting chlorophyll a/b protein (LHCP) is an approximately 25,000-D thylakoid membrane protein. LHCP is synthesized in the cytosol as a precursor and must translocate across the chloroplast envelope before becoming integrally associated with the thylakoid bilayer. Previous studies demonstrated that imported LHCP traverses the chloroplast stroma as a soluble intermediate before thylakoid insertion. Here, examination of this intermediate revealed that it is a stable, discrete approximately 120,000-D species and thus either an LHCP oligomer or a complex with another component. In vitro-synthesized LHCP can be converted to a similar form by incubation with a stromal extract. The stromal component responsible for this conversion is proteinaceous as evidenced by its inactivation by heat, protease, and NEM. Furthermore, the conversion activity coelutes from a gel filtration column with a stromal protein factor(s) previously shown to be necessary for LHCP integration into isolated thylakoids. Conversion of LHCP to the 120-kD form prevents aggregation and maintains its competence for thylakoid insertion. However, conversion to this form is apparently not sufficient for membrane insertion because the isolated 120-kD LHCP still requires stroma to complete the integration process. This suggests a need for at least one more stroma-mediated reaction. Our results explain how a hydrophobic thylakoid protein remains soluble as it traverses the aqueous stroma. Moreover, they describe in part the function of the stromal requirement for insertion into the thylakoid membrane.

Chlorophyll↗

PubMed1991

Molecular cloning and nucleotide sequencing of the coat protein gene of citrus tristeza virus.

Citrus tristeza virus (CTV) contains approximately 20,000 bases of positive-sense ssRNA, encapsidated by a coat protein of approximately 25,000 Mr that has previously been reported to consist of at least two size variants, cp1 and cp2. In the present study, a cDNA library of the T36 isolate of CTV was prepared in a protein expression vector and screened with a polyclonal antibody against the coat protein. Five immunopositive clones produced proteins in Escherichia coli that reacted with monoclonal as well as polyclonal antibodies to the CTV coat protein. Nucleotide sequence analysis of a region common to the five clones revealed the presence of a 669 nucleotide open reading frame flanked by numerous in-frame termination codons. The encoded protein has a predicted Mr of 24,909 and an amino acid composition consistent with that previously reported for the CTV coat protein. Comparison of the predicted amino acid sequence of the coat protein with the amino-terminal sequences of cp1 and cp2 indicated that these coat protein species arise from the same primary translation product, as a result of post-translational proteolysis at sites approximately 12 to 15 and 26 amino acids from the amino terminus respectively. These results are the first reported cloning and sequencing of a CTV gene and provide evidence that CTV may be translated using subgenomic RNA.

Amino Acid Sequence↗

PubMed1991

Cryopreservation of chloroplasts and thylakoids for studies of protein import and integration.

A method is presented for preservation of isolated intact chloroplasts and isolated thylakoids for use in chloroplast protein import and thylakoid protein integration studies. Chloroplasts of pea (Pisum sativum) were preserved by storage in liquid nitrogen in the presence of a cryoprotective agent. Dimethyl sulfoxide was the most effective of several cryoprotectants examined. Approximately 65 to 70% of chloroplasts stored in liquid nitrogen in the presence of dimethyl sulfoxide remained intact upon thawing and were fully functional for the import of precursor proteins. Imported proteins were correctly localized within these chloroplasts, a process that for two of the proteins tested involved transport into the thylakoids. Lysate obtained from preserved chloroplasts was functional for protein integration assays. Preserved chloroplasts retained import and localization capability for up to 6 months of storage. Thylakoids were preserved by a modification of a method previously described (Farkas DL, Malkin S [1979] Plant Physiol 64: 942-947) for preservation of photosynthetic competence. Preserved thylakoids were nearly as active for protein integration studies as freshly prepared thylakoids. The ability to store chloroplasts and subfractions for extended periods will facilitate investigations of plastid protein biogenesis.

Journal Article↗

PubMed1990

Early events in the import/assembly pathway of an integral thylakoid protein.

The light-harvesting chlorophyll a/b protein (LHCP) is nuclear-encoded and must traverse the chloroplast envelope before becoming integrally assembled into thylakoid membranes. Previous studies implicated a soluble stromal form of LHCP in the assembly pathway, but relied upon assays in which the thylakoid insertion step was intentionally impaired [Cline, K., Fulsom, D. R. and Viitanen, P. V. (1989) J. Biol. Chem. 264, 14225-14232]. Here we have developed a rapid-stopping procedure, based upon the use of HgCl2, to analyze early events of the uninhibited assembly process. With this approach, we have found that proper assembly of LHCP into thylakoids lags considerably behind trans-envelope translocation. During the first few minutes of import, two distinct populations of mature-size LHCP accumulate within the chloroplast. One is the aforementioned soluble stromal intermediate, while the other is a partially (or improperly) assembled thylakoid species. Consistent with precursor/product relationships, both species reach peak levels at a time when virtually none of the imported molecules are correctly assembled. These results confirm and extend our previous interpretation, that upon import, preLHCP is rapidly processed to its mature form, giving rise to a soluble stromal intermediate. They further suggest that the stromal intermediate initially inserts into the thylakoid bilayer in a partially assembled form, which eventually becomes properly assembled into the light-harvesting complex.

Biological Transport↗

PubMed1989

An imported thylakoid protein accumulates in the stroma when insertion into thylakoids is inhibited.

The light-harvesting chlorophyll a/b protein (LHCP) is synthesized in the cytosol as a precursor (pLHCP) that is imported into chloroplasts and assembled into thylakoid membranes. Under appropriate conditions, either pLHCP or LHCP will integrate into isolated thylakoids. We have identified two situations that inhibit integration in this assay. Ionophores and uncouplers inhibited integration up to 70%. Carboxyl-terminal truncations of pLHCP also interfered with integration. A 22-residue truncation reduced integration to about 25% of control, whereas a 93 residue truncation completely abolished it. When pLHCP was imported into chloroplasts in the presence of uncouplers or when truncated forms of pLHCP were used, significant amounts of the imported proteins failed to insert into thylakoids and instead accumulated in the aqueous stroma. Accumulation of stromal LHCP occurred at uncoupler concentrations required to dissipate the trans-thylakoid proton electrochemical gradient and was enhanced at reduced levels of ATP. The latter effect may be a secondary consequence of a reduction in ATP-dependent degradation within the stroma. These results indicate that the stroma is an intermediate location in the LHCP assembly pathway and provide the first evidence for a soluble intermediate during biogenesis of a chloroplast membrane protein.

Adenosine Triphosphate↗

PubMed1988

Light-Harvesting Chlorophyll a/b Protein : Membrane Insertion, Proteolytic Processing, Assembly into LHC II, and Localization to Appressed Membranes Occurs in Chloroplast Lysates.

The apoprotein of the light-harvesting chlorophyll a/b protein (LHCP) is a major integral thylakoid membrane protein that is normally complexed with chlorophyll and xanthophylls and serves as the antenna complex of photosystem II. LHCP is encoded in the nucleus and synthesized in the cytosol as a higher molecular weight precursor that is subsequently imported into chloroplasts and assembled into thylakoids. In a previous study it was established that the LHCP precursor can integrate into isolated thylakoid membranes. The present study demonstrates that under conditions designed to preserve thylakoid structure, the inserted LHCP precursor is processed to mature size, assembled into the LHC II chlorophyll-protein complex, and localized to the appressed thylakoid membranes. Under these conditions, light can partially replace exogenous ATP in the membrane integration process.

Journal Article↗

PubMed1988

A Soluble Protein Factor is Required in Vitro for Membrane Insertion of the Thylakoid Precursor Protein, pLHCP.

The precursor to the light-harvesting chlorophyll a/b protein of photosystem II can insert into isolated thylakoid membranes if reaction mixtures also contain ATP and a soluble extract of chloroplasts. Optimization of this insertion process and the initial characterization of the soluble chloroplastic component are presented. With a fixed amount of precursor, maximum integration rates occurred during the first 30 minutes at pH 8.0 and 30 degrees C when the soluble chloroplast extract was increased eight-fold over the stoichiometric amount. Under these conditions, insertion was routinely about 60% of that which occurred during import into intact chloroplasts. Integration also increased virtually linearly with increasing amounts of precursor. However, assays revealed that at least 40% of the in vitro-synthesized pLHCP was pelletable and inactive. The soluble chloroplastic component exhibited characteristics expected of a protein. It was inactivated by heat, protease, and N-ethylmaleimide, but was insensitive to ribonuclease. The soluble component migrated on a Sephacryl S-200 gel filtration column as a single peak with an M(r) of approximately 65,000. The proteinaceous nature of this factor suggests a similarity to soluble factors required for protein transport/integration in other membrane systems.

Journal Article↗