The turnover of brain proteins is critical for organism survival, and its perturbations are linked to pathology. Nevertheless, protein lifetimes have been difficult to obtain in vivo. They are readily measured in vitro by feeding cells with isotopically labeled amino acids, followed by mass spectrometry analyses. In vivo proteins are generated from at least two sources: labeled amino acids from the diet, and non-labeled amino acids from the degradation of pre-existing proteins. This renders measurements difficult. Here we solved this problem rigorously with a workflow that combines mouse in vivo isotopic labeling, mass spectrometry, and mathematical modeling. We also established several independent approaches to test and validate the results. This enabled us to measure the accurate lifetimes of ~3500 brain proteins. The high precision of our data provided a large set of biologically significant observations, including pathway-, organelle-, organ-, or cell-specific effects, along with a comprehensive catalog of extremely long-lived proteins (ELLPs).

Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P 2 ] occurs in the apical plasma membrane of growing pollen tubes. Because enzymes responsible for PtdIns(4,5)P 2 production at that location are uncharacterized, functions of PtdIns(4,5)P 2 in pollen tube tip growth are unresolved. Two candidate genes encoding pollen-expressed Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinases (PI4P 5-kinases) of Arabidopsis subfamily B were identified (PIP5K4 and PIP5K5), and their recombinant proteins were characterized as being PI4P 5-kinases. Pollen of T-DNA insertion lines deficient in both PIP5K4 and PIP5K5 exhibited reduced pollen germination and defects in pollen tube elongation. Fluorescence-tagged PIP5K4 and PIP5K5 localized to an apical plasma membrane microdomain in Arabidopsis and tobacco (Nicotiana tabacum) pollen tubes, and overexpression of either PIP5K4 or PIP5K5 triggered multiple tip branching events. Further studies using the tobacco system revealed that overexpression caused massive apical pectin deposition accompanied by plasma membrane invaginations. By contrast, callose deposition and cytoskeletal structures were unaltered in the overexpressors. Morphological effects depended on PtdIns(4,5)P 2 production, as an inactive enzyme variant did not produce any effects. The data indicate that excessive PtdIns(4,5)P 2 production by type B PI4P 5-kinases disturbs the balance of membrane trafficking and apical pectin deposition. Polar tip growth of pollen tubes may thus be modulated by PtdIns(4,5)P 2 via regulatory effects on membrane trafficking and/or apical pectin deposition.

Root hairs are extensions of root epidermal cells and a model system for directional tip growth of plant cells. A previously uncharacterized Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinase gene (PIP5K3) was identified and found to be expressed in the root cortex, epidermal cells, and root hairs. Recombinant PIP5K3 protein was catalytically active and converted phosphatidylinositol-4-phosphate to phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P 2 ]. Arabidopsis mutant plants homozygous for T-DNA-disrupted PIP5K3 alleles were compromised in root hair formation, a phenotype complemented by expression of wild-type PIP5K3 cDNA under the control of a 1500-bp PIP5K3 promoter fragment. Root hair-specific PIP5K3 overexpression resulted in root hair deformation and loss of cell polarity with increasing accumulation of PIP5K3 transcript. Using reestablishment of root hair formation in T-DNA mutants as a bioassay for physiological functionality of engineered PIP5K3 variants, catalytic activity was found to be essential for physiological function, indicating that PtdIns(4,5)P 2 formation is required for root hair development. An N-terminal domain containing membrane occupation and recognition nexus repeats, which is not required for catalytic activity, was found to be essential for the establishment of root hair growth. Fluorescencetagged PIP5K3 localized to the periphery of the apical region of root hair cells, possibly associating with the plasma membrane and/or exocytotic vesicles. Transient heterologous expression of full-length PIP5K3 in tobacco (Nicotiana tabacum) pollen tubes increased plasma membrane association of a PtdIns(4,5)P 2 -specific reporter in these tip-growing cells. The data demonstrate that root hair development requires PIP5K3-dependent PtdIns(4,5)P 2 production in the apical region of root hair cells.

Chlorophyll is the most abundant photosynthetic pigment in higher plants. During senescence, chlorophyll is hydrolyzed, resulting in the release of free phytol and chlorophyllide. Although the degradation of chlorophyllide has been studied in depth, the metabolic fate of phytol in plants is less clear. Here, we provide evidence that phytol can be incorporated into chlorophyll, tocopherol, and lipid esters by Arabidopsis seedlings. Phytol is phosphorylated to phytyl-phosphate and phytyl-diphosphate by two successive kinase activities associated with chloroplast envelope membranes of Arabidopsis. Although phytol kinase is CTP-dependent, the second kinase reaction, phytyl-phosphate kinase, shows broader specificity for CTP, GTP, UTP, and ATP. Therefore, in addition to de novo synthesis from geranylgeranyl-diphosphate, phosphorylation of free phytol represents an alternative route for phytyl-diphosphate production as the precursor for chloroplast prenyl lipid synthesis. Lipid esters are produced after feeding phytol to Arabidopsis seedlings, and they also accumulate in large amounts in leaves during senescence. The predominant phytyl ester that accumulates during senescence is hexadecatrienoic acid phytyl ester. Fatty acid phytyl ester synthesis by protein extracts of Arabidopsis is stimulated in the presence of phytol-and acyl-CoA esters. Thus, Arabidopsis contains a distinct enzymatic machinery for redirecting free phytol released from chlorophyll degradation into chloroplast lipid metabolism.Isoprenoids represent one of the most diverse classes of naturally occurring compounds. In plants, photosynthetic pigments (i.e. carotenoids and chlorophyll) are derived from isoprenoid biosynthesis. Furthermore, electron carriers of photosynthesis (plastoquinone, phylloquinone), respiration (ubiquinone), and antioxidants (tocopherol) contain isoprenyl side chains (1). Two pathways for isoprenoid synthesis exist in higher plants, the mevalonate pathway localized to the cytosol and the methylerythritol-phosphate pathway found in plastids (2, 3). Plastid isoprenoid metabolism largely depends on the methylerythritol-phosphate pathway. However, some exchange of isoprenoid units between the plastid and the cytosol seems to occur (4). Geranylgeranyl-diphosphate plays an important role in plastid isoprenoid metabolism, because it is the precursor for the synthesis of carotenoids, tocotrienols, chlorophyll, and phytyl-diphosphate (phytyl-PP).3 The existence of two pathways for chlorophyll synthesis was suggested. The first is the direct transfer of a phytyl group onto chlorophyllide from phytyl-PP at the envelope membrane, and the second is the geranylgeranylation of chlorophyllide at the thylakoid membranes (5). Keller et al. (6) identified an Arabidopsis cDNA encoding geranylgeranyl reductase. This enzyme was proposed to convert geranylgeranyl-diphosphate into phytyl-PP and, in addition, to reduce the geranylgeranylated form of chlorophyll to (phytyl-)chlorophyll. A large fraction of phytyl-PP is channeled into chlorophyll synthe...

The repair of inflamed, demyelinated lesions as in multiple sclerosis necessitates the clearance of cholesterol-rich myelin debris by microglia/macrophages and the switch from a pro-inflammatory to an anti-inflammatory lesion environment. Subsequently, oligodendrocytes increase cholesterol levels as a prerequisite for synthesizing new myelin membranes. We hypothesized that lesion resolution is regulated by the fate of cholesterol from damaged myelin combined with oligodendroglial sterol synthesis. By integrating expression profiling, genetics, and comprehensive phenotyping, we found that paradoxically sterol synthesis in myelin-phagocytosing microglia/macrophages determines repair of acutely demyelinated lesions. Rather than producing cholesterol, microglia/macrophages synthesized desmosterol, the immediate cholesterol precursor. Desmosterol activated LXR-signaling to resolve inflammation, creating a permissive environment for oligodendrocyte differentiation. Moreover, LXR-target gene products facilitated the efflux of lipid/cholesterol from lipid-laden microglia/macrophages to support remyelination by oligodendrocytes. Consequently, pharmacological stimulation of sterol synthesis boosted repair of demyelinated lesions, suggesting novel therapeutic strategies for myelin repair in multiple sclerosis.