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)P2] occurs in the apical plasma membrane of growing pollen tubes. Because enzymes responsible for PtdIns(4,5)P2 production at that location are uncharacterized, functions of PtdIns(4,5)P2 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)P2 production, as an inactive enzyme variant did not produce any effects. The data indicate that excessive PtdIns(4,5)P2 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)P2 via regulatory effects on membrane trafficking and/or apical pectin deposition.

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.

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)P2]. 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)P2 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. Fluorescence-tagged 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)P2-specific reporter in these tip-growing cells. The data demonstrate that root hair development requires PIP5K3-dependent PtdIns(4,5)P2 production in the apical region of root hair cells.

The functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] during vegetative plant growth remain obscure. Here, we targeted two related phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) PIP5K1 and PIP5K2, which are expressed ubiquitously in Arabidopsis thaliana. A pip5k1 pip5k2 double mutant with reduced PtdIns(4,5)P₂ levels showed dwarf stature and phenotypes suggesting defects in auxin distribution. The roots of the pip5k1 pip5k2 double mutant had normal auxin levels but reduced auxin transport and altered distribution. Fluorescence-tagged auxin efflux carriers PIN-FORMED (PIN1)–green fluorescent protein (GFP) and PIN2-GFP displayed abnormal, partially apolar distribution. Furthermore, fewer brefeldin A–induced endosomal bodies decorated by PIN1-GFP or PIN2-GFP formed in pip5k1 pip5k2 mutants. Inducible overexpressor lines for PIP5K1 or PIP5K2 also exhibited phenotypes indicating misregulation of auxin-dependent processes, and immunolocalization showed reduced membrane association of PIN1 and PIN2. PIN cycling and polarization require clathrin-mediated endocytosis and labeled clathrin light chain also displayed altered localization patterns in the pip5k1 pip5k2 double mutant, consistent with a role for PtdIns(4,5)P₂ in the regulation of clathrin-mediated endocytosis. Further biochemical tests on subcellular fractions enriched for clathrin-coated vesicles (CCVs) indicated that pip5k1 and pip5k2 mutants have reduced CCV-associated PI4P 5-kinase activity. Together, the data indicate an important role for PtdIns(4,5)P₂ in the control of clathrin dynamics and in auxin distribution in Arabidopsis.

Understanding cellular life requires a comprehensive knowledge of the essential cellular functions, the components involved, and their interactions. Minimized genomes are an important tool to gain this knowledge. We have constructed strains of the model bacterium, Bacillus subtilis, whose genomes have been reduced by ∼36%. These strains are fully viable, and their growth rates in complex medium are comparable to those of wild type strains. An in-depth multi-omics analysis of the genome reduced strains revealed how the deletions affect the transcription regulatory network of the cell, translation resource allocation, and metabolism. A comparison of gene counts and resource allocation demonstrates drastic differences in the two parameters, with 50% of the genes using as little as 10% of translation capacity, whereas the 6% essential genes require 57% of the translation resources. Taken together, the results are a valuable resource on gene dispensability in B. subtilis, and they suggest the roads to further genome reduction to approach the final aim of a minimal cell in which all functions are understood.