The Sll0606 Protein Is Required for Photosystem II Assembly/Stability in the Cyanobacterium Synechocystis sp. PCC 6803

An insertional transposon mutation in the sll0606 gene was found to lead to a loss of photoautotrophy but not photoheterotrophy in the cyanobacterium Synechocystis sp. PCC 6803. Complementation analysis of this mutant (Tsll0606) indicated that an intact sll0606 gene could fully restore photoautotrophic growth. Gene organization in the vicinity of sll0606 indicates that it is not contained in an operon. No electron transport activity was detected in Tsll0606 using water as an electron donor and 2,6-dichlorobenzoquinone as an electron acceptor, indicating that Photosystem II (PS II) was defective. Electron transport activity using dichlorophenol indolephenol plus ascorbate as an electron donor to methyl viologen, however, was the same as observed in the control strain. This indicated that electron flow through Photosystem I was normal. Fluorescence induction and decay parameters verified that Photosystem II was highly compromised. The quantum yield for energy trapping by Photosystem II (F_V/F_M) in the mutant was less than 10% of that observed in the control strain. The small variable fluorescence yield observed after a single saturating flash exhibited aberrant Q_A⁻ reoxidation kinetics that were insensitive to dichloromethylurea. Immunological analysis indicated that whereas the D2 and CP47 proteins were modestly affected, the D1 and CP43 components were dramatically reduced. Analysis of two-dimensional blue native/lithium dodecyl sulfate-polyacrylamide gels indicated that no intact PS II monomer or dimers were observed in the mutant. The CP43-less PS II monomer did accumulate to detectable levels. Our results indicate that the Sll0606 protein is required for the assembly/stability of a functionally competent Photosystem II.     

A chloroplast membrane lacking Photosystem II. Thylakoid stacking in the absence of the Photosystem II particle

The polypeptide composition and membrane structure of a variegated mutant of tobacco have been investigated. The pale green mutant leaf regions contain chloroplasts in which the amount of membrane stacking has been reduced (although not totally eliminated). The mutant membranes are almost totally deficient in Photosystem II when compared to wild-type chloroplast membranes, but still show near-normal levels of Photosystem I activity. The pattern of membrane polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows several differences between mutant and wild-type membranes, although the major chlorophyll-protein complexes described in many other plant species are present in both mutant and wild-type samples. Freeze-fracture analysis of the internal structure of these photosynthetic membranes shows that the Photosystem II-deficient membranes lack the characteristic large particle associated with the E fracture face of the thylakoid. These membranes also lack a tetramer-like particle visible on the inner (ES) surface of the membrane. The other characteristics of the photosynthetic membrane, including the small particles observed on the P fracture faces in both stacked and unstacked regions, and the characteristic changes in the background matrix of the E fracture face which accompany thylakoid stacking, are unaltered in the mutant. From these and other observations we conclude that the large (EF and ES) particle represents an amalgam of many components comprising the Photosystem II reaction complex, that the absence of one or more of its components may prevent the structure from assembling, and that in its absence, Photosystem II activity cannot be observed.     

The PsbP Domain Protein 1 Functions in the Assembly of Lumenal Domains in Photosystem I

Photosystem I (PS I) is a multisubunit membrane protein complex that functions as a light-driven plastocyanin-ferredoxin oxidoreductase. The PsbP domain protein 1 (PPD1; At4g15510) is located in the thylakoid lumen of plant chloroplasts and is essential for photoautotrophy, functioning as a PS I assembly factor. In this work, RNAi was used to suppress PPD1 expression, yielding mutants displaying a range of phenotypes with respect to PS I accumulation and function. These PPD1 RNAi mutants showed a loss of assembled PS I that was correlated with loss of the PPD1 protein. In the most severely affected PPD1 RNAi lines, the accumulated PS I complexes exhibited defects in electron transfer from plastocyanin to the oxidized reaction center P₇₀₀⁺. The defects in PS I assembly in the PPD1 RNAi mutants also had secondary effects with respect to the association of light-harvesting antenna complexes to PS I. Because of the imbalance in photosystem function in the PPD1 RNAi mutants, light-harvesting complex II associated with and acted as an antenna for the PS I complexes. These results provide new evidence for the role of PPD1 in PS I biogenesis, particularly as a factor essential for proper assembly of the lumenal portion of the complex.     

A Conserved Rubredoxin Is Necessary for Photosystem II Accumulation in Diverse Oxygenic Photoautotrophs

In oxygenic photosynthesis, two photosystems work in tandem to harvest light energy and generate NADPH and ATP. Photosystem II (PSII), the protein-pigment complex that uses light energy to catalyze the splitting of water, is assembled from its component parts in a tightly regulated process that requires a number of assembly factors. The 2pac mutant of the unicellular green alga Chlamydomonas reinhardtii was isolated and found to have no detectable PSII activity, whereas other components of the photosynthetic electron transport chain, including photosystem I, were still functional. PSII activity was fully restored by complementation with the RBD1 gene, which encodes a small iron-sulfur protein known as a rubredoxin. Phylogenetic evidence supports the hypothesis that this rubredoxin and its orthologs are unique to oxygenic phototrophs and distinct from rubredoxins in Archaea and bacteria (excluding cyanobacteria). Knockouts of the rubredoxin orthologs in the cyanobacterium Synechocystis sp. PCC 6803 and the plant Arabidopsis thaliana were also found to be specifically affected in PSII accumulation. Taken together, our data suggest that this rubredoxin is necessary for normal PSII activity in a diverse set of organisms that perform oxygenic photosynthesis.     

Cysteine 70 of Ankyrin-G Is S-Palmitoylated and Is Required for Function of Ankyrin-G in Membrane Domain Assembly

Ankyrin-G (AnkG) coordinates protein composition of diverse membrane domains, including epithelial lateral membranes and neuronal axon initial segments. However, how AnkG itself localizes to these membrane domains is not understood. We report that AnkG remains on the plasma membrane in Madin-Darby canine kidney (MDCK) cells grown in low calcium, although these cells lack apical-basal polarity and exhibit loss of plasma membrane association of AnkG partners, E-cadherin and β₂-spectrin. We subsequently demonstrate using mutagenesis and mass spectrometry that AnkG is S-palmitoylated exclusively at Cys-70, which is located in a loop of the first ankyrin repeat and is conserved in the vertebrate ankyrin family. Moreover, C70A mutation abolishes membrane association of 190-kDa AnkG in MDCK cells grown in low calcium. C70A 190-kDa AnkG fails to restore biogenesis of epithelial lateral membranes in MDCK cells depleted of endogenous AnkG. In addition, C70A 270-kDa AnkG fails to cluster at the axon initial segment of AnkG-depleted cultured hippocampal neurons and fails to recruit neurofascin as well as voltage-gated sodium channels. These effects of C70A mutation combined with evidence for its S-palmitoylation are consistent with a requirement of palmitoylation for targeting and function of AnkG in membrane domain biogenesis at epithelial lateral membranes and neuronal axon initial segments.     

A Functional DnaK Dimer Is Essential for the Efficient Interaction with Hsp40 Heat Shock Protein

Highly conserved molecular chaperone Hsp70 heat shock proteins play a key role in maintaining protein homeostasis (proteostasis). DnaK, a major Hsp70 in Escherichia coli, has been widely used as a paradigm for studying Hsp70s. In the absence of ATP, purified DnaK forms low-ordered oligomer, whereas ATP binding shifts the equilibrium toward the monomer. Recently, we solved the crystal structure of DnaK in complex with ATP. There are two molecules of DnaK-ATP in the asymmetric unit. Interestingly, the interfaces between the two molecules of DnaK are large with good surface complementarity, suggesting functional importance of this crystallographic dimer. Biochemical analyses of DnaK protein supported the formation of dimer in solution. Furthermore, our cross-linking experiment based on the DnaK-ATP structure confirmed that DnaK forms specific dimer in an ATP-dependent manner. To understand the physiological function of the dimer, we mutated five residues on the dimer interface. Four mutations, R56A, T301A, N537A, and D540A, resulted in loss of chaperone activity and compromised the formation of dimer, indicating the functional importance of the dimer. Surprisingly, neither the intrinsic biochemical activities, the ATP-induced allosteric coupling, nor GrpE co-chaperone interaction is affected appreciably in all of the mutations except for R56A. Unexpectedly, the interaction with co-chaperone Hsp40 is significantly compromised. In summary, this study suggests that DnaK forms a transient dimer upon ATP binding, and this dimer is essential for the efficient interaction of DnaK with Hsp40.     

A chloroplast membrane lacking Photosystem I. Changes in unstacked membrane regions

The structure and polypeptide composition of the photosynthetic membrane of a mutant of maize has been investigated. The thylakoid membranes of the mutant plants are deficient in Photosystem I activity, although Photosystem II is at near normal levels. SDS polyacrylamide gel electrophoresis of thylakoid membranes from the mutant shows them to be deficient in two polypeptide bands which have been associated with Photosystem I. Freeze-fracture studies of the membrane show that the absence of these polypeptides is associated with a measurable reduction in particle diameter on the unstacked protoplasmic fracture face. This fracture face is derived from the splitting of membranes in unstacked regions of the thylakoid membrane system. It is suggested that in membranes stacked by salts in vitro, Photosystem I activity may be confined to this region.     

Identification of the Chromophores Involved in Aggregation-dependent Energy Quenching of the Monomeric Photosystem II Antenna Protein Lhcb5

Non-photochemical quenching (NPQ) of excess absorbed light energy is a fundamental process that regulates photosynthetic light harvesting in higher plants. Among several proposed NPQ mechanisms, aggregation-dependent quenching (ADQ) and charge transfer quenching have received the most attention. In vitro spectroscopic features of both mechanisms correlate with very similar signals detected in more intact systems and in vivo, where full NPQ can be observed. A major difference between the models is the proposed quenching site, which is predominantly the major trimeric light-harvesting complex II in ADQ and exclusively monomeric Lhcb proteins in charge transfer quenching. Here, we studied ADQ in both monomeric and trimeric Lhcb proteins, investigating the activities of each antenna subunit and their dependence on zeaxanthin, a major modulator of NPQ in vivo. We found that monomeric Lhcb proteins undergo stronger quenching than light-harvesting complex II during aggregation and that this is enhanced by binding to zeaxanthin, as occurs during NPQ in vivo. Finally, the analysis of Lhcb5 mutants showed that chlorophyll 612 and 613, in close contact with lutein bound at site L1, are important facilitators of ADQ.     

A Membrane-located Glycosyltransferase Complex Required for Biosynthesis of the d-Galactan I Lipopolysaccharide O Antigen in Klebsiella pneumoniae

d-Galactan I is a polysaccharide with the disaccharide repeat unit structure [→3-β-d-Galf-(1→3)-α-d-Galp-(1→]. This glycan represents the lipopolysaccharide O antigen found in many Gram-negative bacteria, including several Klebsiella pneumoniae O serotypes. The polysaccharide is synthesized in the cytoplasm prior to its export via an ATP-binding cassette transporter. Sequence analysis predicts three galactosyltransferases in the d-galactan I genetic locus. They are WbbO (belonging to glycosyltransferase (GT) family 4), WbbM (GT-family 8), and WbbN (GT-family 2). The WbbO and WbbM proteins are each predicted to contain two domains, with the GT modules located toward their C termini. The N-terminal domains of WbbO and WbbM exhibit no similarity to proteins with known function. In vivo complementation assays suggest that all three glycosyltransferases are required for d-galactan I biosynthesis. Using a bacterial two-hybrid system and confirmatory co-purification strategies, evidence is provided for protein-protein interactions among the glycosyltransferases, creating a membrane-located enzyme complex dedicated to d-galactan I biosynthesis.     

Fat Storage-inducing Transmembrane Protein 2 Is Required for Normal Fat Storage in Adipose Tissue

Triglycerides within the cytosol of cells are stored in a phylogenetically conserved organelle called the lipid droplet (LD). LDs can be formed at the endoplasmic reticulum, but mechanisms that regulate the formation of LDs are incompletely understood. Adipose tissue has a high capacity to form lipid droplets and store triglycerides. Fat storage-inducing transmembrane protein 2 (FITM2/FIT2) is highly expressed in adipocytes, and data indicate that FIT2 has an important role in the formation of LDs in cells, but whether FIT2 has a physiological role in triglyceride storage in adipose tissue remains unproven. Here we show that adipose-specific deficiency of FIT2 (AF2KO) in mice results in progressive lipodystrophy of white adipose depots and metabolic dysfunction. In contrast, interscapular brown adipose tissue of AF2KO mice accumulated few but large LDs without changes in cellular triglyceride levels. High fat feeding of AF2KO mice or AF2KO mice on the genetically obese ob/ob background accelerated the onset of lipodystrophy. At the cellular level, primary adipocyte precursors of white and brown adipose tissue differentiated in vitro produced fewer but larger LDs without changes in total cellular triglyceride or triglyceride biosynthesis. These data support the conclusion that FIT2 plays an essential, physiological role in fat storage in vivo.     

Outer Membrane Protein I of Pseudomonas aeruginosa Is a Target of Cationic Antimicrobial Peptide/Protein

Cationic antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms against invading microorganisms. Here we demonstrate that OprI (outer membrane protein I) of Pseudomonas aeruginosa is responsible for its susceptibility to human ribonuclease 7 (hRNase 7) and α-helical cationic AMPs, instead of surface lipopolysaccharide, which is the initial binding site of cationic AMPs. The antimicrobial activities of hRNase 7 and α-helical cationic AMPs against P. aeruginosa were inhibited by the addition of exogenous OprI or anti-OprI antibody. On modification and internalization of OprI by hRNase 7 into cytosol, the bacterial membrane became permeable to metabolites. The lipoprotein was predicted to consist of an extended loop at the N terminus for hRNase 7/lipopolysaccharide binding, a trimeric α-helix, and a lysine residue at the C terminus for cell wall anchoring. Our findings highlight a novel mechanism of antimicrobial activity and document a previously unexplored target of α-helical cationic AMPs, which may be used for screening drugs to treat antibiotic-resistant bacterial infection.     

Structure and Functional Analysis of LptC, a Conserved Membrane Protein Involved in the Lipopolysaccharide Export Pathway in Escherichia coli

LptC is a conserved bitopic inner membrane protein from Escherichia coli involved in the export of lipopolysaccharide from its site of synthesis in the cytoplasmic membrane to the outer membrane. LptC forms a complex with the ATP-binding cassette transporter, LptBFG, which is thought to facilitate the extraction of lipopolysaccharide from the inner membrane and release it into a translocation pathway that includes the putative periplasmic chaperone LptA. Cysteine modification experiments established that the catalytic domain of LptC is oriented toward the periplasm. The structure of the periplasmic domain is described at a resolution of 2.2-Å from x-ray crystallographic data. The periplasmic domain of LptC consists of a twisted boat structure with two β-sheets in apposition to each other. The β-sheets contain seven and eight antiparallel β-strands, respectively. This structure bears a high degree of resemblance to the crystal structure of LptA. Like LptA, LptC binds lipopolysaccharide in vitro. In vitro, LptA can displace lipopolysaccharide from LptC (but not vice versa), consistent with their locations and their proposed placement in a unidirectional export pathway.     

The Ubiquitin C-Terminal Hydrolase L1 (UCH-L1) C Terminus Plays a Key Role in Protein Stability,but Its Farnesylation Is Not Required for Membrane Association in Primary Neurons

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is highly expressed in neurons. A possible role for UCH-L1 in neurodegeneration has been highlighted because of its presence in Lewy bodies associated with Parkinson disease and neurofibrillary tangles observed in Alzheimer disease. UCH-L1 exists in two forms in neurons, a soluble cytoplasmic form (UCH-L1^C) and a membrane-associated form (UCH-L1^M). Alzheimer brains show reduced levels of soluble UCH-L1^C correlating with the formation of UCH-L1-immunoreactive tau tangles, whereas UCH-L1^M has been implicated in α-synuclein dysfunction. Given these reports of divergent roles, we investigated the properties of UCH-L1 membrane association. Surprisingly, our results indicate that UCH-L1 does not partition to the membrane in the cultured cell lines we tested. Furthermore, in primary cultured neurons, a proportion of UCH-L1^M does partition to the membrane, but, contrary to a previous report, this does not require farnesylation. Deletion of the four C-terminal residues caused the loss of protein solubility, abrogation of substrate binding, increased cell death, and an abnormal intracellular distribution, consistent with protein dysfunction and aggregation. These data indicate that UCH-L1 is differently processed in neurons compared with clonal cell lines and that farnesylation does not account for the membrane association in neurons.     

Structure of Transmembrane Domain of Lysosome-associated Membrane Protein Type 2a (LAMP-2A) Reveals Key Features for Substrate Specificity in Chaperone-mediated Autophagy

Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation.     

Trm112p Is a 15-kDa Zinc Finger Protein Essential for the Activity of Two tRNA and One Protein Methyltransferases in Yeast

The degenerate base at position 34 of the tRNA anticodon is the target of numerous modification enzymes. In Saccharomyces cerevisiae, five tRNAs exhibit a complex modification of uridine 34 (mcm⁵U₃₄ and mcm⁵s²U₃₄), the formation of which requires at least 25 different proteins. The addition of the last methyl group is catalyzed by the methyltransferase Trm9p. Trm9p interacts with Trm112p, a 15-kDa protein with a zinc finger domain. Trm112p is essential for the activity of Trm11p, another tRNA methyltransferase, and for Mtq2p, an enzyme that methylates the translation termination factor eRF1/Sup45. Here, we report that Trm112p is required in vivo for the formation of mcm⁵U₃₄ and mcm⁵s²U₃₄. When produced in Escherichia coli, Trm112p forms a complex with Trm9p, which renders the latter soluble. This recombinant complex catalyzes the formation of mcm⁵U₃₄ on tRNA in vitro but not mcm⁵s²U₃₄. An mtq2-0 trm9-0 strain exhibits a synthetic growth defect, thus revealing the existence of an unexpected link between tRNA anticodon modification and termination of translation. Trm112p is associated with other partners involved in ribosome biogenesis and chromatin remodeling, suggesting that it has additional roles in the cell.     

A mechanism for the formation of inside-out membrane vesicles. Preparation of inside-out vesicles from membrane-paired randomized chloroplast lamellae

Inside-out thylakoid membrane vesicles can be isolated by aqueous polymer two-phase partition of Yeda press-fragmented spinach chloroplasts (Andersson, B. and Åkerlund, H.-E. (1978) Biochim. Biophys. Acta 503, 462–472). The mechanism for their formation has been investigated by studying the yield of inside-out vesicles after various treatments of the chloroplasts prior to fragmentation. No inside-out vesicles were isolated during phase partitioning if the chloroplasts had been destacked in a low-salt medium prior to the fragmentation. Only in those cases where the chloroplast lamellae had been stacked by cations or membrane-paired by acidic treatment did we get any yield of inside-out vesicles. Thus, the intrinsic properties of chloroplast thylakoids seem to be such that they seal into right-side out vesicles after disruption unless they are in an appressed state. This favours the following mechanism for the formation of inside-out thylakoids. After press treatment, a ruptured membrane still remains appressed with an adjacent membrane. Resealing of such an appressed membrane pair would result in an inside-out vesicle.If the compartmentation of chloroplast lamellae into appressed grana and unappressed stroma lamellae is preserved by cations before fragmentation, the inside-out vesicles are highly enriched in photosystem II. This indicates a granal origin which is consistent with the proposed model outlined. Inside-out vesicles possessing photosystem I and II properties in approximately equal proportions could be obtained by acid-induced membrane-pairing of chloroplasts which had been destacked and randomized prior to fragmentation. Since this new preparation of inside-out thylakoid vesicles also exposes components derived from the stroma lamellae it complements the previous preparation.It is suggested that fragmentation of paired membranes followed by phase partitioning should be a general method of obtaining inside-out vesicles from membranes of various biological sources.     

A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and its interaction with the human chaperone cyclophilin A are both targets for highly potent and promising antiviral drugs that are in the late stages of clinical development. Despite its high interest in regards to the development of drugs to counteract the worldwide HCV burden, NS5A is still an enigmatic multifunctional protein poorly characterized at the molecular level. NS5A is required for HCV RNA replication and is involved in viral particle formation and regulation of host pathways. Thus far, no enzymatic activity or precise molecular function has been ascribed to NS5A that is composed of a highly structured domain 1 (D1), as well as two intrinsically disordered domains 2 (D2) and 3 (D3), representing half of the protein. Here, we identify a short structural motif in the disordered NS5A-D2 and report its NMR structure. We show that this structural motif, a minimal Pro³¹⁴–Trp³¹⁶ turn, is essential for HCV RNA replication, and its disruption alters the subcellular distribution of NS5A. We demonstrate that this Pro-Trp turn is required for proper interaction with the host cyclophilin A and influences its peptidyl-prolyl cis/trans isomerase activity on residue Pro³¹⁴ of NS5A-D2. This work provides a molecular basis for further understanding of the function of the intrinsically disordered domain 2 of HCV NS5A protein. In addition, our work highlights how very small structural motifs present in intrinsically disordered proteins can exert a specific function.