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1. Proteomics of isolated sieve tubes from Nicotiana tabacum : sieve element–specific proteins reveal differentiation of the endomembrane system

   https://doi.org/10.1073/pnas.2112755119  

   Liu, Yan ; Vasina, Viktoriya V. ; Kraner, Max E. ; Peters, Winfried S. ; Sonnewald, Uwe ; Knoblauch, Michael ( January 2022 , Proceedings of the National Academy of Sciences)

   Symplasmicly connected cells called sieve elements form a network of tubes in the phloem of vascular plants. Sieve elements have essential functions as they provide routes for photoassimilate distribution, the exchange of developmental signals, and the coordination of defense responses. Nonetheless, they are the least understood main type of plant cells. They are extremely sensitive, possess a reduced endomembrane system without Golgi apparatus, and lack nuclei and translation machineries, so that transcriptomics and similar techniques cannot be applied. Moreover, the analysis of phloem exudates as a proxy for sieve element composition is marred by methodological problems. We developed a simple protocol for the isolation of sieve elements from leaves and stems of Nicotiana tabacum at sufficient amounts for large-scale proteome analysis. By quantifying the enrichment of individual proteins in purified sieve element relative to bulk phloem preparations, proteins of increased likelyhood to function specifically in sieve elements were identified. To evaluate the validity of this approach, yellow fluorescent protein constructs of genes encoding three of the candidate proteins were expressed in plants. Tagged proteins occurred exclusively in sieve elements. Two of them, a putative cytochrome b561/ferric reductase and a reticulon-like protein, appeared restricted to segments of the endoplasmic reticulum (ER)more »that were inaccessible to green fluorescent protein dissolved in the ER lumen, suggesting a previously unknown differentiation of the endomembrane system in sieve elements. Evidently, our list of promising candidate proteins ( SI Appendix , Table S1 ) provides a valuable exploratory tool for sieve element biology.« less
2. Reaction of O ₂ with a diiron protein generates a mixed-valent Fe ²⁺ /Fe ³⁺ center and peroxide

   https://doi.org/10.1073/pnas.1809913116  

   Bradley, Justin M. ; Svistunenko, Dimitri A. ; Pullin, Jacob ; Hill, Natalie ; Stuart, Rhona K. ; Palenik, Brian ; Wilson, Michael T. ; Hemmings, Andrew M. ; Moore, Geoffrey R. ; Le Brun, Nick E. ( February 2019 , Proceedings of the National Academy of Sciences)

   The gene encoding the cyanobacterial ferritinSynFtn is up-regulated in response to copper stress. Here, we show that, whileSynFtn does not interact directly with copper, it is highly unusual in several ways. First, its catalytic diiron ferroxidase center is unlike those of all other characterized prokaryotic ferritins and instead resembles an animal H-chain ferritin center. Second, as demonstrated by kinetic, spectroscopic, and high-resolution X-ray crystallographic data, reaction of O₂with the di-Fe²⁺center results in a direct, one-electron oxidation to a mixed-valent Fe²⁺/Fe³⁺form. Iron–O₂chemistry of this type is currently unknown among the growing family of proteins that bind a diiron site within a four α-helical bundle in general and ferritins in particular. The mixed-valent form, which slowly oxidized to the more usual di-Fe³⁺form, is an intermediate that is continually generated during mineralization. Peroxide, rather than superoxide, is shown to be the product of O₂reduction, implying that ferroxidase centers function in pairs via long-range electron transfer through the protein resulting in reduction of O₂bound at only one of the centers. We show that electron transfer is mediated by the transient formation of a radical on Tyr40, which lies ∼4 Å from the diiron center. As well as demonstrating an expansion of the iron–O₂chemistry knownmore »to occur in nature, these data are also highly relevant to the question of whether all ferritins mineralize iron via a common mechanism, providing unequivocal proof that they do not.

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3. The γ-tubulin complex protein GCP6 is crucial for spindle morphogenesis but not essential for microtubule reorganization in Arabidopsis

   https://doi.org/10.1073/pnas.1912240116  

   Miao, Huiying ; Guo, Rongfang ; Chen, Junlin ; Wang, Qiaomei ; Lee, Yuh-Ru Julie ; Liu, Bo ( December 2019 , Proceedings of the National Academy of Sciences)

   γ-Tubulin typically forms a ring-shaped complex with 5 related γ-tubulin complex proteins (GCP2 to GCP6), and this γ-tubulin ring complex (γTuRC) serves as a template for microtubule (MT) nucleation in plants and animals. While the γTuRC takes part in MT nucleation in most eukaryotes, in fungi such events take place robustly with just the γ-tubulin small complex (γTuSC) assembled by γ-tubulin plus GCP2 and GCP3. To explore whether the γTuRC is the sole functional γ-tubulin complex in plants, we generated 2 mutants of theGCP6gene encoding the largest subunit of the γTuRC inArabidopsis thaliana. Both mutants showed similar phenotypes of dwarfed vegetative growth and reduced fertility. Thegcp6mutant assembled the γTuSC, while the wild-type cells had GCP6 join other GCPs to produce the γTuRC. Although thegcp6cells had greatly diminished γ-tubulin localization on spindle MTs, the protein was still detected there. Thegcp6cells formed spindles that lacked MT convergence and discernable poles; however, they managed to cope with the challenge of MT disorganization and were able to complete mitosis and cytokinesis. Our results reveal that the γTuRC is not the only functional form of the γ-tubulin complex for MT nucleation in plant cells, and that γ-tubulin-dependent, but γTuRC-independent, mechanisms meet the basal need ofmore »MT nucleation. Moreover, we show that the γTuRC function is more critical for the assembly of spindle MT array than for the phragmoplast. Thus, our findings provide insight into acentrosomal MT nucleation and organization.

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4. Initial Characterization of the Two ClpP Paralogs of Chlamydia trachomatis Suggests Unique Functionality for Each

   https://doi.org/10.1128/JB.00635-18  

   Wood, Nicholas A. ; Chung, Krystal Y. ; Blocker, Amanda M. ; Rodrigues de Almeida, Nathalia ; Conda-Sheridan, Martin ; Fisher, Derek J. ; Ouellette, Scot P. ; Stock, Ann M. ( January 2019 , Journal of Bacteriology)

   ABSTRACT Members of Chlamydia are obligate intracellular bacteria that differentiate between two distinct functional and morphological forms during their developmental cycle, elementary bodies (EBs) and reticulate bodies (RBs). EBs are nondividing small electron-dense forms that infect host cells. RBs are larger noninfectious replicative forms that develop within a membrane-bound vesicle, termed an inclusion. Given the unique properties of each developmental form of this bacterium, we hypothesized that the Clp protease system plays an integral role in proteomic turnover by degrading specific proteins from one developmental form or the other. Chlamydia spp. have five uncharacterized clp genes, clpX , clpC , two clpP paralogs, and clpB . In other bacteria, ClpC and ClpX are ATPases that unfold and feed proteins into the ClpP protease to be degraded, and ClpB is a deaggregase. Here, we focused on characterizing the ClpP paralogs. Transcriptional analyses and immunoblotting determined that these genes are expressed midcycle. Bioinformatic analyses of these proteins identified key residues important for activity. Overexpression of inactive clpP mutants in Chlamydia spp. suggested independent function of each ClpP paralog. To further probe these differences, we determined interactions between the ClpP proteins using bacterial two-hybrid assays and native gel analysis of recombinant proteins. Homotypicmore »interactions of the ClpP proteins, but not heterotypic interactions between the ClpP paralogs, were detected. Interestingly, protease activity of ClpP2, but not ClpP1, was detected in vitro . This activity was stimulated by antibiotics known to activate ClpP, which also blocked chlamydial growth. Our data suggest the chlamydial ClpP paralogs likely serve distinct and critical roles in this important pathogen. IMPORTANCE Chlamydia trachomatis is the leading cause of preventable infectious blindness and of bacterial sexually transmitted infections worldwide. Chlamydiae are developmentally regulated obligate intracellular pathogens that alternate between two functional and morphologic forms, with distinct repertoires of proteins. We hypothesize that protein degradation is a critical aspect to the developmental cycle. A key system involved in protein turnover in bacteria is the Clp protease system. Here, we characterized the two chlamydial ClpP paralogs by examining their expression in Chlamydia spp., their ability to oligomerize, and their proteolytic activity. This work will help understand the evolutionarily diverse Clp proteases in the context of intracellular organisms, which may aid in the study of other clinically relevant intracellular bacteria.« less
5. Sieve-element differentiation and phloem sap contamination

   Michael Knoblauch, Winfried S. ( January 2018 , Current opinion in plant biology)

   Sieve elements (SEs) degrade selected organelles and cytoplasmic structures when they differentiate. According to classical investigations, only smooth ER, mitochondria, sieve element plastids, and, in most cases, P-proteins remain in mature SEs. More recent proteomics and immunohistochemical studies, however, suggested that additional components including a protein-synthesizing machinery and a fully developed actin cytoskeleton operate in mature SEs. These interpretations are at odds with conventional imaging studies. Here we discuss potential causes for these discrepancies, concluding that differentiating SEs may play a role by ‘contaminating’ phloem exudates.