Phosphorylation of Photosystem II Controls Functional Macroscopic Folding of Photosynthetic Membranes in Arabidopsis

Photosynthetic thylakoid membranes in plants contain highly folded membrane layers enriched in photosystem II, which uses light energy to oxidize water and produce oxygen. The sunlight also causes quantitative phosphorylation of major photosystem II proteins. Analysis of the Arabidopsis thaliana stn7xstn8 double mutant deficient in thylakoid protein kinases STN7 and STN8 revealed light-independent phosphorylation of PsbH protein and greatly reduced N-terminal phosphorylation of D2 protein. The stn7xstn8 and stn8 mutants deficient in light-induced phosphorylation of photosystem II had increased thylakoid membrane folding compared with wild-type and stn7 plants. Significant enhancement in the size of stacked thylakoid membranes in stn7xstn8 and stn8 accelerated gravity-driven sedimentation of isolated thylakoids and was observed directly in plant leaves by transmission electron microscopy. Increased membrane folding, caused by the loss of light-induced protein phosphorylation, obstructed lateral migration of the photosystem II reaction center protein D1 and of processing protease FtsH between the stacked and unstacked membrane domains, suppressing turnover of damaged D1 in the leaves exposed to high light. These findings show that the high level of photosystem II phosphorylation in plants is required for adjustment of macroscopic folding of large photosynthetic membranes modulating lateral mobility of membrane proteins and sustained photosynthetic activity.The use of captured sunlight energy to split water and drive oxygenic photosynthesis by photosystem II (PSII) (Barber, 2006) inevitably generates reactive oxygen species and causes oxidative damage to the PSII protein pigment complex. The light-induced damage to PSII, in particular to the D1 reaction center protein, requires PSII repair to sustain its photosynthetic function (Takahashi and Murata, 2008). Impairment and degradation of D1 increase with rising light intensities, and this protein has the fastest turnover rate among the photosynthetic proteins of plants, algae, and cyanobacteria (Yokthongwattana and Melis, 2006). However, in plants, the PSII is segregated in highly stacked membrane layers of very large thylakoid membranes (Andersson and Anderson, 1980; Kirchhoff et al., 2008), which are densely folded to fit inside chloroplasts (Mullineaux, 2005; Shimoni et al., 2005). As a consequence, the PSII repair cycle in plants is slower than in cyanobacteria (Yokthongwattana and Melis, 2006), and it includes migration of the PSII complex from the stacked membrane domains (grana) to the unstacked membranes (stroma lamellae), where proteolysis and insertion of a newly synthesized D1 protein occurs (Baena-Gonzalez and Aro, 2002; Yokthongwattana and Melis, 2006). High light also causes quantitative phosphorylation of the membrane surface–exposed regions of D1, D2, CP43, and PsbH proteins of PSII in plants (Rintamäki et al., 1997; Vener et al., 2001), but the function of this phosphorylation is largely unknown and reports on its importance for the D1 protein turnover are conflicting (Bonardi et al., 2005; Tikkanen et al., 2008).Phosphorylation of the PSII proteins in Arabidopsis thaliana depends mostly on the light-activated protein kinase STN8 (Vainonen et al., 2005), while the STN7 kinase is essential for phosphorylation of the light-harvesting proteins of PSII (Bellafiore et al., 2005; Bonardi et al., 2005; Tikkanen et al., 2006). An earlier study on Arabidopsis mutants lacking both STN7 and STN8 (stn7xstn8), as well as only STN8, concluded that protein phosphorylation was not essential for PSII repair (Bonardi et al., 2005), while more recent work revealed a dramatic retardation in D1 degradation under high light in the stn8 and stn7xstn8 mutants (Tikkanen et al., 2008). Moreover, it was shown that the lack of PSII phosphorylation resulted in accumulation of photodamaged PSII complexes and in general oxidative damage of photosynthetic proteins in the thylakoid membranes under high light (Tikkanen et al., 2008). The other study revealed that the stn7xstn8 double mutant grown under natural field conditions produced 41% less seeds than wild-type plants (Frenkel et al., 2007), which also indicated physiological importance of thylakoid protein phosphorylation in maintenance of plant fitness.To uncover the function of light-dependent protein phosphorylation in plant photosynthetic membranes, we performed a detailed analysis of the Arabidopsis mutants deficient in the protein kinases STN7 and STN8. The earlier published results on protein phosphorylation analyses in the stn7xstn8 mutant of Arabidopsis were restricted to antiphosphothreonine antibody-based immunodetection and did not reveal any phosphorylation of PSII core proteins (Bonardi et al., 2005; Tikkanen et al., 2008). Using a mass spectrometry (MS) approach and immunoblot analyses with two complementary antiphosphothreonine antibodies, we find remaining light-independent phosphorylation of PsbH and D2 proteins of PSII in stn7xstn8. We demonstrate that degradation and aggregation patterns of the D1 protein in stn7xstn8 differ from those in wild-type, stn7, and stn8 plants. We also observe a reproducible delay in the degradation of D1 in high light–treated leaves of stn7xstn8 and stn8 compared with the wild-type and stn7 plants. Finally, we show that phosphorylation of PSII proteins modulates macroscopic rearrangements of the entire membrane network of plant thylakoids, which facilitates lateral mobility of membrane proteins, required for repair and sustained activity of PSII.     

Profound redox sensitivity of peptidyl-prolyl isomerase activity in Arabidopsis thylakoid lumen

Proteomic, enzymatic, and mutant analyses revealed that peptidyl-prolyl isomerase (PPIase) activity in the chloroplast thylakoid lumen of Arabidopsis is determined by two immunophilins: AtCYP20-2 and AtFKBP13. These two enzymes are responsible for PPIase activity in both soluble and membrane-associated fractions of thylakoid lumen suggesting that other lumenal immunophilins are not active towards the peptide substrates. In thiol-reducing conditions PPIase activity of the isolated AtFKBP13 and of the total thylakoid lumen is suppressed several fold. Profound redox-dependence of PPIase activity implies oxidative activation of protein folding catalysis under oxidative stress and photosynthetic oxygen production in the thylakoid lumen of plant chloroplasts.     

Selective growth of normal adult human urothelial cells in serum-free medium

Summary A serum-free medium (HMRI-2) has been developed for the outgrowth and subculture of epithelial cells from normal adult human ureter and bladder. Medium HMRI-2 consists of Ham’s MCDB 152 with double the amounts of the essential amino acids in Stock 1, low Ca²⁺ (0.06 mM) and is supplemented with epithelial growth factor, 5 ng/ml; transferrin, 5 μg/ml; insulin, 5 μg/ml; ethanolamine and phosphoethanolamine, 0.1 mM each; hydrocortisone, 2.8×10⁻⁶ M; and bovine pituitary extract, 126 μg protein/ml. The cultured cells showed ultrastructural markers of epithelial cells (prekeratin fibers, tonofilaments, surface microvilli with glycocalyx), exhibited ABO antigens, and had a normal human diploid karyotype. Primary cultures could be subcultured and also cryopreserved in HMRI-2 in liquid nitrogen. Cells in mass cultures showed a population doubling time of 40.5±4.5 h and had a maximum in vitro life span of 20 to 25 population doublings. It was observed that primary outgrowths, secondary cultures, and even cryopreserved cells all retained the capacity to respond to high Ca²⁺ and serum by differentiation and desquamation. This study has resulted in the availability of easily obtainable serum-free epithelial cultures from normal adult human ureter and bladder. The useful in vitro life span of these cultures may be important in future studies of carcinogenesis. This work was supported by a grant from the National Cancer Institute (R01CA25089), Bethesda, MD.     

Light-harvesting complex II protein CP29 binds to photosystem I of Chlamydomonas reinhardtii under State 2 conditions

The State 1 to State 2 transition in the photosynthetic membranes of plants and green algae involves the functional coupling of phosphorylated light-harvesting complexes of photosystem II (LHCII) to photosystem I (PSI). We present evidence suggesting that in Chlamydomonas reinhardtii this coupling may be aided by a hyper-phosphorylated form of the LHCII-like CP29 protein (Lhcbm4). MS analysis of CP29 showed that Thr6, Thr16 and Thr32, and Ser102 are phosphorylated in State 2, whereas in State 1-exposed cells only phosphorylation of Thr6 and Thr32 could be detected. The LHCI-PSI supercomplex isolated from the alga in State 2 was found to contain strongly associated CP29 in phosphorylated form. Electron microscopy suggests that the binding site for this highly phosphorylated CP29 is close to the PsaH protein. It is therefore postulated that redox-dependent multiple phosphorylation of CP29 in green algae is an integral part of the State transition process in which the structural changes of CP29, induced by reversible phosphorylation, determine the affinity of LHCII for either of the two photosystems.     

Feedback equilibrium control during human standing

Equilibrium maintenance during standing in humans was investigated with a 3-joint (ankle, knee and hip) sagittal model of body movement. The experimental paradigm consisted of sudden perturbations of humans in quiet stance by backward displacements of the support platform. Data analysis was performed using eigenvectors of motion equation. The results supported three conclusions. First, independent feedback control of movements along eigenvectors (eigenmovements) can adequately describe human postural responses to stance perturbations. This conclusion is consistent with previous observations (Alexandrov et al., 2001b) that these same eigenmovements are also independently controlled in a feed-forward manner during voluntary upper-trunk bending. Second, independent feedback control of each eigenmovement is sufficient to provide its stability. Third, the feedback loop in each eigenmovement can be modeled as a linear visco-elastic spring with delay. Visco-elastic parameters and time-delay values result from the combined contribution of passive visco-elastic mechanisms and sensory systems of different modalities     

N-terminal processing and modifications of caveolin-1 in caveolae from human adipocytes

Caveolin, the principal structural protein of caveolae membrane domains, has a cytosol-exposed N-terminal part that was cleaved off by trypsin treatment of caveolae vesicles isolated from primary human adipocytes. Sequencing of the released tryptic peptides by nanospray quadrupole time-of-flight mass spectrometry revealed that both caveolin-1alpha and caveolin-1beta were processed by excision of the starting methionines. The N-terminus of the mature caveolin-1alpha was acetylated, while caveolin-1beta was found in acetylated as well as in non-acetylated forms. Fractional phosphorylation of serine-36 in the mature caveolin-1alpha and of the homologous serine-5 in caveolin-1beta was identified. This is the first experimental evidence for in vivo phosphorylation of caveolin-1 at the consensus site for phosphorylation by protein kinase C. The phosphorylation was found in both the acetylated and non-acetylated variants of caveolin-1beta. This variability in modifications is consistent with critical involvement of the N-terminal domain of caveolin in the regulation of caveolae.     

Analysis of SBIR phase I and phase II review results at the National Institutes of Health

A cohort of phase I and phase II summary statements for the SBIR grant applications was evaluated to determine the strengths and weaknesses in approved and disapproved applications. An analysis of outcome variables (disapproval or unfunded status) was examined with respect to exposure variables (strengths or shortcomings). Logistic regression models were developed for comparisons to measure the predictive value of shortcomings and strengths to the outcomes. Disapproved phase I results were compared with an earlier 1985 study. Although the magnitude of the frequencies of shortcomings was greater in the present study, the relative rankings within shortcoming class were more alike than different. Also, the frequencies of shortcomings were, with one exception, not significantly different in the two studies. Differences in the summary statement review may have accounted for some differences observed between the 1985 data and results of the present study. Comparisons of Approved/Disapproved and Approved-Unfunded/Funded yielded the following observations. For phase I applicants, a lack of a clearly stated, testable hypothesis, a poorly qualified or described investigative team, and inadequate methodological approaches contributed significantly (in that order) to a rating of disapproval. A critical flaw for phase II proposals was failure to accomplish objectives of the phase I study. Methodological issues also dominate the distinctions in both comparison groups. A clear result of the data presented here and that published previously is that SBIR applicants need continuing assistance to improve the chances of their success. These results should serve as a guide to assist NIH staff as they provide information to prospective applicants focusing on key elements of the application. A continuing review of the SBIR program would be helpful to evaluate the quality of the submitted science.     

Use of biotinylated inorganic pyrophosphatase for detection of biotin bound to solid support

A colorimetric procedure to detect biotin bound to microtiter plates with a sensitivity down to 10(-16) mol was developed using biotinylated inorganic pyrophosphatase of Escherichia coli. Reaction of pyrophosphatase with 1 mM N-biotinyl-6-aminocaproic acid N-hydroxy-sulfonosuccinimide ester yielded a stable 87% active enzyme containing 5.6 mol biotin/mol. In the measurements of human immunoglobulin G, a biotinylated pyrophosphatase.streptavidin complex provided a sensitivity superior to that of conventional enzyme immunoassay due to low nonspecific binding. The new procedure was also more sensitive compared with that using biotinylated alkaline phosphatase. Together with high thermostability of pyrophosphatase and its substrate, low background staining allowed measurement of enzymatic activity to be performed at 60 degrees C for 4 h resulting in a marked increase in assay sensitivity.