Interactions of ribosome nascent chain complexes of the chloroplast-encoded D1 thylakoid membrane protein with cpSRP54.

The mechanisms of targeting, insertion and assembly of the chloroplast-encoded thylakoid membrane proteins are unknown. In this study, we investigated these mechanisms for the chloroplast-encoded polytopic D1 thylakoid membrane protein, using a homologous translation system isolated from tobacco chloroplasts. Truncated forms of the psbA gene were translated and stable ribosome nascent chain complexes were purified. To probe the interactions with the soluble components of the targeting machinery, we used UV-activatable cross-linkers incorporated at specific positions in the nascent chains, as well as conventional sulfhydryl cross-linkers. With both cross-linking approaches, the D1 ribosome nascent chain was photocross-linked to cpSRP54. cpSRP54 was shown to interact only when the D1 nascent chain was still attached to the ribosome. The interaction was strongly dependent on the length of the nascent chain that emerged from the ribosome, as well as the cross-link position. No interactions with soluble SecA or cpSRP43 were found. These results imply a role for cpSRP54 in D1 biogenesis.     

A SecY homologue is involved in chloroplast-encoded D1 protein biogenesis

We have used the photosystem II reaction center D1 protein as a model to study the mechanisms of targeting and insertion of chloroplast-encoded thylakoid membrane proteins. The unusually high turnover rate and distinct pausing intermediates during translation make the D1 protein biogenesis particularly suitable for these purposes. Here we show that cpSecY, a chloroplast homologue of bacterial essential translocon component SecY, interacts tightly with thylakoid membrane-bound ribosomes, suggesting its involvement in protein translocation and insertion. Co-immunoprecipitation and cross-linking experiments indicated that cpSecY resides in the vicinity of D1 elongation intermediates and provided evidence for a transient interaction of cpSecY with D1 elongation intermediates during the biogenesis of D1. After termination of translation, such interactions no longer existed. Our results indicate that, in addition to a well characterized role of cpSecY in posttranslational translocation of nuclear-encoded proteins, it seems to be also involved in cotranslational membrane protein translocation and insertion in chloroplasts.     

The role of chloroplast-encoded protein biosynthesis on the rate of D1 protein degradation in Dunaliella salina

Mechanistic aspects of the Photosystem II (PS II) damage and repair cycle in Dunaliella salina were investigated. The work addressed the role of chloroplast-encoded protein biosynthesis on the rate of the D1 protein (chloroplast psbA gene product) degradation, following photoinhibition of PS II under in vivo conditions. Cells were grown under different light-intensities and the rate of D1 photodamage and degradation was measured via pulse-chase measurements with (³⁵S)sulfate. It is shown that no detectable difference exists in the rate of D1 degradation in D. salina, measured in the presence or absence of lincomycin, a chloroplast protein biosynthesis inhibitor. The results suggest that de novo D1 biosynthesis does not play a role in the regulation of D1 degradation. In low-light (100 mol photons m^–2 s^–1) grown cells, the rate of photodamage to D1 did not exceed the rate of its degradation and replacement. In high-light (2200 mol photons m^–1 s^–1) grown cells, the rate of D1 photodamage was faster than the rate of its degradation, resulting in a significant accumulation of photoinactivated PS II centers in the chloroplast thylakoids (chronic photoinhibition). The latter was coincident with the appearance of a 160 kD complex that contained photodamaged D1. Electron micrographs of D. salina thylakoids revealed extensive grana stacks in the thylakoid membrane of low-light grown cells. Only rudimentary appressions consisting of simple membrane pairings were found in the high-light grown cells. The results are discussed in terms of the regulation of D1 degradation in chloroplasts under in vivo conditions.Abbreviations Chl chlorophyll - D1 the 32 kD reaction center protein of PS II, encoded by the chloroplast psbA gene - D2 the 34 kD reaction center protein of PS II, encoded by the chloroplast psbD gene - HL high light - LL low light - Linc lincomycin     

A conserved motif is prerequisite for the interaction of NAC with ribosomal protein L23 and nascent chains

In eukaryotes, newly synthesized proteins interact co-translationally with a multitude of different ribosome-bound factors and chaperones including the conserved heterodimeric nascent polypeptide-associated complex (NAC) and a Hsp40/70-based chaperone system. These factors are thought to play an important role in protein folding and targeting, yet their specific ribosomal localizations, which are prerequisite for their functions, remain elusive. This study describes the ribosomal localization of NAC and the molecular details by which NAC is able to contact the ribosome and gain access to nascent polypeptides. We identified a conserved RRK(X)nKK ribosome binding motif within the beta-subunit of NAC that is essential for the entire NAC complex to attach to ribosomes and allow for its interaction with nascent polypeptide chains. The motif localizes within a potential loop region between two predicted alpha-helices in the N terminus of betaNAC. This N-terminal betaNAC ribosome-binding domain was completely portable and sufficient to target an otherwise cytosolic protein to the ribosome. NAC modified with a UV-activatable cross-linker within its ribosome binding motif specifically cross-linked to L23 ribosomal protein family members at the exit site of the ribosome, providing the first evidence of NAC-L23 interaction in the context of the ribosome. Mutations of L23 reduced NAC ribosome binding in vivo and in vitro, whereas other eukaryotic ribosome-associated factors such as the Hsp70/40 chaperones Ssb or Zuotin were unaffected. We conclude that NAC employs a conserved ribosome binding domain to position itself on the L23 ribosomal protein adjacent to the nascent polypeptide exit site.     

Mapping the interaction of the STT3 subunit of the oligosaccharyl transferase complex with nascent polypeptide chains

Many secretory and membrane proteins are N-glycosylated by the oligosaccharyl transferase complex during their translocation across the endoplasmic reticulum membrane. Several experimental observations suggest that the highly conserved STT3 subunit contains the active site of the oligosaccharyl transferase. Here, we report a detailed study of the interaction between the active site of the STT3 protein and nascent polypeptide chains using an in vitro photocrosslinking technique. Our results show that the addition of a glycan moiety in a stretch of approximately 15 residues surrounding a QK(*)T cross-linking site impairs the interaction between the nascent chain and STT3.     

Biogenesis of the chloroplast-encoded D1 protein: regulation of translation elongation, insertion, and assembly into photosystem II

Regulation of translation elongation, membrane insertion, and assembly of the chloroplast-encoded D1 protein of photosystem II (PSII) was studied using a chloroplast translation system in organello. Translation elongation of D1 protein was found to be regulated by (1) a redox component that can be activated not only by photosynthetic electron transfer but also by reduction with DTT; (2) the trans-thylakoid proton gradient, which is absolutely required for elongation of D1 nascent chains on the thylakoid membrane; and (3) the thiol reactants N-ethylmaleimide (NEM) and iodosobenzoic acid (IBZ), which inhibit translation elongation with concomitant accumulation of distinct D1 pausing intermediates. These results demonstrate that D1 translation elongation and membrane insertion are tightly coupled and highly regulated processes in that proper insertion is a prerequisite for translation elongation of D1. Cotranslational and post-translational assembly steps of D1 into PSII reaction center and core complexes occurred independently of photosynthetic electron transfer or trans-thylakoid proton gradient but were strongly affected by the thiol reactants DTT, NEM, and IBZ. These compounds reduced the stability of the early PSII assembly intermediates, hampered the C-terminal processing of the precursor of D1, and prevented the post-translational reassociation of CP43, indicating a strong dependence of the D1 assembly steps on proper redox conditions and the formation of disulfide bonds.     

Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas

A rat monoclonal antibody specific for immunoglobulin (Ig) heavy chain binding protein (BiP) has allowed the examination of the association of BiP with assembling Ig precursors in mouse B lymphocyte-derived cell lines. The anti-BiP monoclonal antibody immunoprecipitates BiP along with noncovalently associated Ig heavy chains. BiP is a component of the endoplasmic reticulum and binds free intracellular heavy chains in nonsecreting pre-B (mu+, L-) cell lines or incompletely assembled Ig precursors in (H+, L+) secreting hybridomas and myelomas. In the absence of light chain synthesis, heavy chains remain associated with BiP and are not secreted. The association of BiP with assembling Ig molecules in secreting hybridomas is transient and is restricted to the incompletely assembled molecules which are found in the endoplasmic reticulum. BiP loses affinity and disassociates with Ig molecules when polymerization with light chain is complete. We propose that the association of BiP with Ig heavy chain precursors is a novel posttranslational processing event occurring in the endoplasmic reticulum. The Ig heavy chains associated with BiP are not efficiently transported from the endoplasmic reticulum to the Golgi apparatus. Therefore, BiP may prevent the premature escape and eventual secretion of incompletely assembled Ig molecules.     

Sec-dependent membrane protein insertion: sequential interaction of nascent FtsQ with SecY and YidC

Recent studies identified YidC as a novel membrane factor that may play a key role in membrane insertion of inner membrane proteins (IMPs), both in conjunction with the Sec-translocase and as a separate entity. Here, we show that the type II IMP FtsQ requires both the translocase and, to a lesser extent, YidC in vivo. Using photo-crosslinking we demonstrate that the transmembrane (TM) domain of the nascent IMP FtsQ inserts into the membrane close to SecY and lipids, and moves to a combined YidC/lipid environment upon elongation. These data are consistent with a crucial role for YidC in the lateral transfer of TM domains from the Sec translocase into the lipid bilayer.     

Initial interaction of apoA-I with ABCA1 impacts in vivo metabolic fate of nascent HDL

We investigated the in vivo metabolic fate of pre-beta HDL particles in human apolipoprotein A-I transgenic (hA-I (Tg)) mice. Pre-beta HDL tracers were assembled by incubation of [(125)I]tyramine cellobiose-labeled apolipoprotein A-I (apoA-I) with HEK293 cells expressing ABCA1. Radiolabeled pre-beta HDLs of increasing size (pre-beta1, -2, -3, and -4 HDLs) were isolated by fast-protein liquid chromatography and injected into hA-I (Tg)-recipient mice, after which plasma decay, in vivo remodeling, and tissue uptake were monitored. Pre-beta2, -3, and -4 had similar plasma die-away rates, whereas pre-beta1 HDL was removed 7-fold more rapidly. Radiolabel recovered in liver and kidney 24 h after tracer injection suggested increased (P < 0.001) liver and decreased kidney catabolism as pre-beta HDL size increased. In plasma, pre-beta1 and -2 were rapidly (<5 min) remodeled into larger HDLs, whereas pre-beta3 and -4 were remodeled into smaller HDLs. Pre-beta HDLs were similarly remodeled in vitro with control or LCAT-immunodepleted plasma, but not when incubated with phospholipid transfer protein knockout plasma. Our results suggest that initial interaction of apoA-I with ABCA1 imparts a unique conformation that partially determines the in vivo metabolic fate of apoA-I, resulting in increased liver and decreased kidney catabolism as pre-beta HDL particle size increases.     

D1 and D2 dopamine receptor expression is regulated by direct interaction with the chaperone protein calnexin

As for all proteins, G protein-coupled receptors (GPCRs) undergo synthesis and maturation within the endoplasmic reticulum (ER). The mechanisms involved in the biogenesis and trafficking of GPCRs from the ER to the cell surface are poorly understood, but they may involve interactions with other proteins. We have now identified the ER chaperone protein calnexin as an interacting protein for both D(1) and D(2) dopamine receptors. These protein-protein interactions were confirmed using Western blot analysis and co-immunoprecipitation experiments. To determine the influence of calnexin on receptor expression, we conducted assays in HEK293T cells using a variety of calnexin-modifying conditions. Inhibition of glycosylation either through receptor mutations or treatments with glycosylation inhibitors partially blocks the interactions with calnexin with a resulting decrease in cell surface receptor expression. Confocal fluorescence microscopy reveals the accumulation of D(1)-green fluorescent protein and D(2)-yellow fluorescent protein receptors within internal stores following treatment with calnexin inhibitors. Overexpression of calnexin also results in a marked decrease in both D(1) and D(2) receptor expression. This is likely because of an increase in ER retention because confocal microscopy revealed intracellular clustering of dopamine receptors that were co-localized with an ER marker protein. Additionally, we show that calnexin interacts with the receptors via two distinct mechanisms, glycan-dependent and glycan-independent, which may underlie the multiple effects (ER retention and surface trafficking) of calnexin on receptor expression. Our data suggest that optimal receptor-calnexin interactions critically regulate D(1) and D(2) receptor trafficking and expression at the cell surface, a mechanism likely to be of importance for many GPCRs.     

Nanoscale elongating control of the self‐assembled protein filament with the cysteine‐introduced building blocks

Self‐assembly of artificially designed proteins is extremely desirable for nanomaterials. Here we show a novel strategy for the creation of self‐assembling proteins, named “Nanolego.” Nanolego consists of “structural elements” of a structurally stable symmetrical homo‐oligomeric protein and “binding elements,” which are multiple heterointeraction proteins with relatively weak affinity. We have established two key technologies for Nanolego, a stabilization method and a method for terminating the self‐assembly process. The stabilization method is mediated by disulfide bonds between Cysteine‐residues incorporated into the binding elements, and the termination method uses “capping Nanolegos,” in which some of the binding elements in the Nanolego are absent for the self‐assembled ends. With these technologies, we successfully constructed timing‐controlled and size‐regulated filament‐shape complexes via Nanolego self‐assembly. The Nanolego concept and these technologies should pave the way for regulated nanoarchitecture using designed proteins.     

Cotranslational protein integration into the ER membrane is mediated by the binding of nascent chains to translocon proteins

During cotranslational protein integration into the ER membrane, each transmembrane (TM) segment moves laterally through the translocon to reach the lipid bilayer. Photocrosslinking studies reveal that a particular surface of each nascent chain TM alpha helix and signal-anchor sequence always faces Sec61alpha in the translocon. This nonrandom and TM sequence-dependent positioning reveals that each TM segment makes specific contacts with Sec61alpha and is retained at a fixed location within the translocon, observations that are best explained by the binding of each TM sequence to a translocon protein(s). Since TM sequence hydrophobicity does not correlate with its rate of release from the translocon, nascent chain movement through the translocon appears to be mediated primarily by protein-protein interactions rather than hydrophobic nascent chain-phospholipid interactions.     

Identification of interaction sites of protein kinase Calpha on phospholipase D1

Phospholipase D (PLD) is regulated by many factors, including protein kinase C (PKC) and small G-proteins of the Rho and ADP-ribosylation factor families. Previous studies revealed that the activation of PLD1 by phorbol ester is associated with the binding of PKCalpha to a site in the N-terminus of PLD1. The purpose of the present study was to determine this site more precisely. Immunoprecipitation with a series of four PLD1 deletion mutants confirmed that PKCalpha strongly interacted with the amino acid sequence 1-318 at the N-terminus of PLD1 and weakly with the sequence 841-1036 at the C-terminus. Further immunoprecipitation studies with deletion mutants of the 1-318 and 1-215 PLD1 fragments revealed that there were binding sites in the 1-49 N-terminal sequence and also in the 216-318 sequence containing the PH domain. Studies of N-terminal deletion mutants of full-length PLD1 confirmed the presence of a binding site in the 1-49 sequence and a further site in the 1-318 sequence. Both deletion mutants showed impaired activation by PKCalpha in vivo, but unchanged activation by active V(14)RhoA. These findings identify the 1-49 sequence is a major binding/activation site for PKCalpha on PLD1, but also indicate involvement of the PH domain.     

N-linked oligosaccharide chains of Sendai virus fusion protein determine the interaction with endoplasmic reticulum molecular chaperones

The selectivity and individual roles of the N-linked oligosaccharide chains of Sendai virus fusion protein (F protein) in the interaction with endoplasmic reticulum molecular chaperones were investigated by analyses of transient expression of single N-glycosylation mutants and sequential immunoprecipitation. We demonstrated differential interactions depending on the location of the N-linked oligosaccharide chain, and showed that these interactions were correlated with the folding and transport of F proteins. Moreover, mutant F proteins that lacked the specific N-linked oligosaccharide chains required for disulfide bond formation showed increased association with ERp57.