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.Abbreviations EPSP 5-enolpyruvyulshikimate-3-phosphate - LHCP Chlorophyll a/b binding protein of the light-harvesting complex - NPT-II Neomycin phosphotransferase II - PC Plastocyanin - Pr Precursor - Rubisco Ribulose-1,5,-bisphosphate carboxylase/oxygenase - SS Small subunit of Rubisco     

Complex interactions between the chaperonin 60 molecular chaperone and dihydrofolate reductase.

The spontaneous refolding of chemically denatured dihydrofolate reductase (DHFR) is completely arrested by chaperonin 60 (GroEL). This inhibition presumably results from the formation of a stable complex between chaperonin 60 and one or more intermediates in the folding pathway. While sequestered on chaperonin 60, DHFR is considerably more sensitive to proteolysis, suggesting a nonnative structure. Bound DHFR can be released from chaperonin 60 with ATP, and although chaperonin 10 (GroES) is not obligatory, it does potentiate the maximum effect of ATP. Hydrolysis of ATP is also not required for DHFR release since certain nonhydrolyzable analogues are capable of partial discharge. "Native" DHFR can also form a stable complex with chaperonin 60. However, in this case, complex formation is not instantaneous and can be prevented by the presence of DHFR substrates. This suggests that native DHFR exists in equilibrium with at least one conformer which is recognizable by chaperonin 60. Binding studies with 35S-labeled DHFR support these conclusions and further demonstrate that DHFR competes for a common saturable site with another protein (ribulose-1,5-bisphosphate carboxylase) known to interact with chaperonin 60.     

Production of sterigmatocystin by Aspergillus versicolor and Bipolaris sorokiniana on semisynthetic liquid and solid media.

Higher yields of sterigmatocystin were obtained with Aspergillus versicolor than with Bipolaris sorokiniana both in liquid and on solid media. The optimum temperature for sterigmatocystin production by A. versicolor was 27 to 29 degrees C and 23 degrees C for B. sorokiniana. In liquid shake cultures, production of sterigmatocystin by B. sorokiniana was negligible, whereas maximal production by A. versicolor was 210 mg/liter. On solid substrates, the highest yields (8 g/kg) were obtained with A. versicolor on still cultures of whole corn supplemented with Soytone.     

Replication slippage may cause parallel evolution in the secondary structures of mitochondrial transfer RNAs

Presence of the dihydrouridine (D) stem in the mitochondrial cysteine tRNA is unusually variable among lepidosaurian reptiles. Phylogenetic and comparative analyses of cysteine tRNA gene sequences identify eight parallel losses of the D-stem, resulting in D-arm replacement loops. Sampling within the monophyletic Acrodonta provides no evidence for reversal. Slipped-strand mispairing of noncontiguous repeated sequences during replication or direct replication slippage can explain repeats observed within cysteine tRNAs that contain a D-arm replacement loop. These two mechanisms involving replication slippage can account for the loss of the cysteine tRNA D-stem in several lepidosaurian lineages, and may represent general mechanisms by which the secondary structures of mitochondrial tRNAs are altered.      

Crystal and molecular structure of polymeric [MnCl2(bpy)]n

A crystal and molecular structure determination of MnCl₂(bpy) showed that it exists as polymeric octahedral [MnCl₂(bpy)]_n. [MnCl₂(bpy)_n crystallizes in the monoclinic space group I2/a with A = 7.007(1), B = 9.200(1), C = 16.495(1) Å, β = 91.313(5)° and Z = 4. On the basis of 979 unique observed reflections with I 2.5σ(I) the structure was refined to R = 0.032.     

cDNA cloning of a developmentally regulated hemocyanin subunit in the crustacean Cancer magister and phylogenetic analysis of the hemocyanin gene family

The complete cDNA sequence and protein reading frame of a developmentally regulated hemocyanin subunit in the Dungeness crab (Cancer magister) is presented. The protein sequence is aligned with 18 potentially homologous hemocyanin-type proteins displaying apparent sequence similarities. Functional domains are identified, and a comparison of predicted hydrophilicities, surface probabilities, and regional backbone flexibilities provides evidence for a remarkable degree of structural conservation among the proteins surveyed. Parsimony analysis of the protein sequence alignment identifies four monophyletic groups on the arthropodan branch of the hemocyanin gene tree: crustacean hemocyanins, insect hexamerins, chelicerate hemocyanins, and arthropodan prophenoloxidases. They form a monophyletic group relative to molluscan hemocyanins and nonarthropodan tyrosinases. Arthropodan prophenoloxidases, although functionally similar to tyrosinases, appear to belong to the arthropodan hexamer- type hemolymph proteins as opposed to molluscan hemocyanins and tyrosinases.      

Several proteins imported into chloroplasts form stable complexes with the GroEL-related chloroplast molecular chaperone.

Nine different proteins were imported into isolated pea chloroplasts in vitro. For seven of these [the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), beta-subunit of ATP synthase, glutamine synthetase, the light-harvesting chlorophyll a/b binding protein, chloramphenicol acetyltransferase, and pre-beta-lactamase], a fraction was found to migrate as a stable high-molecular-weight complex during nondenaturing gel electrophoresis. This complex contained the mature forms of the imported proteins and the groEL-related chloroplast chaperonin 60 (previously known as Rubisco subunit binding protein). Thus, the stable association of imported proteins with this molecular chaperone is widespread and not necessarily restricted to Rubisco subunits or to chloroplast proteins. With two of the imported proteins (ferredoxin and superoxide dismutase), such complexes were not observed. It seems likely that, in addition to its proposed role in assembly of Rubisco, the chloroplast chaperonin 60 is involved in the assembly or folding of a wide range of proteins in chloroplasts.     

Sorting of major cargo glycoproteins into clathrin-coated vesicles

The AP-1 and AP-2 complexes are the most abundant adaptors in clathrin-coated vesicles (CCVs), but clathrin-mediated trafficking can still occur in the absence of any detectable AP-1 or AP-2. To find out whether adaptor abundance reflects cargo abundance, we used lectin pulldowns to identify the major membrane glycoproteins in CCVs from human placenta and rat liver. Both preparations contained three prominent high molecular-weight proteins: the cation-independent mannose 6-phosphate receptor (CIMPR), carboxypeptidase D (CPD) and low-density lipoprotein receptor-related protein 1 (LRP1). To investigate how these proteins are sorted, we constructed and stably transfected CD8 chimeras into HeLa cells. CD8-CIMPR localized mainly to early/tubular endosomes, CD8-CPD to the trans Golgi network and CD8-LRP1 to late/multivesicular endosomes. All three constructs redistributed to the plasma membrane when clathrin was depleted by siRNA. CD8-CIMPR was also strongly affected by AP-2 depletion. CD8-CPD was moderately affected by AP-2 depletion but strongly affected by depleting AP-1 and AP-2 together. CD8-LRP1 was only slightly affected by AP-2 depletion; however, mutating an NPXY motif in the LRP1 tail caused it to become AP-2 dependent. These results indicate that all three proteins have AP-dependent sorting signals, which may help to explain the relative abundance of AP complexes in CCVs. However, the relatively low abundance of cargo proteins in CCV preparations suggests either that some of the APs may be empty or that the preparations may be dominated by empty coats.     

Pyrrolidine-constrained phenethylamines: The design of potent, selective, and pharmacologically efficacious dipeptidyl peptidase IV (DPP4) inhibitors from a lead-like screening hit

A novel series of pyrrolidine-constrained phenethylamines were developed as dipeptidyl peptidase IV (DPP4) inhibitors for the treatment of type 2 diabetes. The cyclohexene ring of lead-like screening hit 5 was replaced with a pyrrolidine to enable parallel chemistry, and protein co-crystal structural data guided the optimization of N-substituents. Employing this strategy, a >400x improvement in potency over the initial hit was realized in rapid fashion. Optimized compounds are potent and selective inhibitors with excellent pharmacokinetic profiles. Compound 30 was efficacious in vivo, lowering blood glucose in ZDF rats that were allowed to feed freely on a mixed meal.