Proteome-scale movements and compartment connectivity during the eukaryotic cell cycle.

Cell cycle progression relies on coordinated changes in the composition and subcellular localization of the proteome. By applying two distinct convolutional neural networks on images of millions of live yeast cells, we resolved proteome-level dynamics in both concentration and localization during the cell cycle, with resolution of ∼20 subcellular localization classes. We show that a quarter of the proteome displays cell cycle periodicity, with proteins tending to be controlled either at the level of localization or concentration, but not both. Distinct levels of protein regulation are preferentially utilized for different aspects of the cell cycle, with changes in protein concentration being mostly involved in cell cycle control and changes in protein localization in the biophysical implementation of the cell cycle program. We present a resource for exploring global proteome dynamics during the cell cycle, which will aid in understanding a fundamental biological process at a systems level.

A chloroplast protein atlas reveals punctate structures and spatial organization of biosynthetic pathways.

Chloroplasts are eukaryotic photosynthetic organelles that drive the global carbon cycle. Despite their importance, our understanding of their protein composition, function, and spatial organization remains limited. Here, we determined the localizations of 1,034 candidate chloroplast proteins using fluorescent protein tagging in the model alga Chlamydomonas reinhardtii. The localizations provide insights into the functions of poorly characterized proteins; identify novel components of nucleoids, plastoglobules, and the pyrenoid; and reveal widespread protein targeting to multiple compartments. We discovered and further characterized cellular organizational features, including eleven chloroplast punctate structures, cytosolic crescent structures, and unexpected spatial distributions of enzymes within the chloroplast. We also used machine learning to predict the localizations of other nuclear-encoded Chlamydomonas proteins. The strains and localization atlas developed here will serve as a resource to accelerate studies of chloroplast architecture and functions.

Systematic identification and characterization of genes in the regulation and biogenesis of photosynthetic machinery.

Photosynthesis is central to food production and the Earth's biogeochemistry, yet the molecular basis for its regulation remains poorly understood. Here, using high-throughput genetics in the model eukaryotic alga Chlamydomonas reinhardtii, we identify with high confidence (false discovery rate [FDR] < 0.11) 70 poorly characterized genes required for photosynthesis. We then enable the functional characterization of these genes by providing a resource of proteomes of mutant strains, each lacking one of these genes. The data allow assignment of 34 genes to the biogenesis or regulation of one or more specific photosynthetic complexes. Further analysis uncovers biogenesis/regulatory roles for at least seven proteins, including five photosystem I mRNA maturation factors, the chloroplast translation factor MTF1, and the master regulator PMR1, which regulates chloroplast genes via nuclear-expressed factors. Our work provides a rich resource identifying regulatory and functional genes and placing them into pathways, thereby opening the door to a system-level understanding of photosynthesis.

Light Controls Protein Localization through Phytochrome-Mediated Alternative Promoter Selection.

Alternative promoter usage is a proteome-expanding mechanism that allows multiple pre-mRNAs to be transcribed from a single gene. The impact of this mechanism on the proteome and whether it is positively exploited in normal organismal responses remain unclear. We found that the plant photoreceptor phytochrome induces genome-wide changes in alternative promoter selection in Arabidopsis thaliana. Through this mechanism, protein isoforms with different N termini are produced that display light-dependent differences in localization. For instance, shade-grown plants accumulate a cytoplasmic isoform of glycerate kinase (GLYK), an essential photorespiration enzyme that was previously thought to localize exclusively to the chloroplast. Cytoplasmic GLYK constitutes a photorespiratory bypass that alleviates fluctuating light-induced photoinhibition. Therefore, phytochrome controls alternative promoter selection to modulate protein localization in response to changing light conditions. This study suggests that alternative promoter usage represents another ubiquitous layer of gene expression regulation in eukaryotes that contributes to diversification of the proteome.

ATP13A1 prevents ERAD of folding-competent mislocalized and misoriented proteins.

The biosynthesis of thousands of proteins requires targeting a signal sequence or transmembrane segment (TM) to the endoplasmic reticulum (ER). These hydrophobic ɑ helices must localize to the appropriate cellular membrane and integrate in the correct topology to maintain a high-fidelity proteome. Here, we show that the P5A-ATPase ATP13A1 prevents the accumulation of mislocalized and misoriented proteins, which are eliminated by different ER-associated degradation (ERAD) pathways in mammalian cells. Without ATP13A1, mitochondrial tail-anchored proteins mislocalize to the ER through the ER membrane protein complex and are cleaved by signal peptide peptidase for ERAD. ATP13A1 also facilitates the topogenesis of a subset of proteins with an N-terminal TM or signal sequence that should insert into the ER membrane with a cytosolic N terminus. Without ATP13A1, such proteins accumulate in the wrong orientation and are targeted for ERAD by distinct ubiquitin ligases. Thus, ATP13A1 prevents ERAD of diverse proteins capable of proper folding.

Co-translational assembly and localized translation of nucleoporins in nuclear pore complex biogenesis.

mRNA translation is coupled to multiprotein complex assembly in the cytoplasm or to protein delivery into intracellular compartments. Here, by combining systematic RNA immunoprecipitation and single-molecule RNA imaging in yeast, we have provided a complete depiction of the co-translational events involved in the biogenesis of a large multiprotein assembly, the nuclear pore complex (NPC). We report that binary interactions between NPC subunits can be established during translation, in the cytoplasm. Strikingly, the nucleoporins Nup1/Nup2, together with a number of nuclear proteins, are instead translated at nuclear pores, through a mechanism involving interactions between their nascent N-termini and nuclear transport receptors. Uncoupling this co-translational recruitment further triggers the formation of cytoplasmic foci of unassembled polypeptides. Altogether, our data reveal that distinct, spatially segregated modes of co-translational interactions foster the ordered assembly of NPC subunits and that localized translation can ensure the proper delivery of proteins to the pore and the nucleus.

SubCellBarCode: Proteome-wide Mapping of Protein Localization and Relocalization.

Subcellular localization is a main determinant of protein function; however, a global view of cellular proteome organization remains relatively unexplored. We have developed a robust mass spectrometry-based analysis pipeline to generate a proteome-wide view of subcellular localization for proteins mapping to 12,418 individual genes across five cell lines. Based on more than 83,000 unique classifications and correlation profiling, we investigate the effect of alternative splicing and protein domains on localization, complex member co-localization, cell-type-specific localization, as well as protein relocalization after growth factor inhibition. Our analysis provides information about the cellular architecture and complexity of the spatial organization of the proteome; we show that the majority of proteins have a single main subcellular location, that alternative splicing rarely affects subcellular location, and that cell types are best distinguished by expression of proteins exposed to the surrounding environment. The resource is freely accessible via www.subcellbarcode.org.

StaufenC facilitates utilization of the ERAD pathway to transport dsRNA through the endoplasmic reticulum to the cytosol.

RNA interference (RNAi) is more efficient in coleopteran insects than other insects. StaufenC (StauC), a coleopteran-specific double-stranded RNA (dsRNA)-binding protein, is required for efficient RNAi in coleopterans. We investigated the function of StauC in the intracellular transport of dsRNA into the cytosol, where dsRNA is digested by Dicer enzymes and recruited by Argonauts to RNA-induced silencing complexes. Confocal microscopy and cellular organelle fractionation studies have shown that dsRNA is trafficked through the endoplasmic reticulum (ER) in coleopteran Colorado potato beetle (CPB) cells. StauC is localized to the ER in CPB cells, and StauC-knockdown caused the accumulation of dsRNA in the ER and a decrease in the cytosol, suggesting that StauC plays a key role in the intracellular transport of dsRNA through the ER. Using immunoprecipitation, we showed that StauC is required for dsRNA interaction with ER proteins in the ER-associated protein degradation (ERAD) pathway, and these interactions are required for RNAi in CPB cells. These results suggest that StauC works with the ERAD pathway to transport dsRNA through the ER to the cytosol. This information could be used to develop dsRNA delivery methods aimed at improving RNAi.

Higher-Order Structural Organization of the Mitochondrial Proteome Charted by In Situ Cross-Linking Mass Spectrometry.

Mitochondria are densely packed with proteins, of which most are involved physically or more transiently in protein-protein interactions (PPIs). Mitochondria host among others all enzymes of the Krebs cycle and the oxidative phosphorylation pathway and are foremost associated with cellular bioenergetics. However, mitochondria are also important contributors to apoptotic cell death and contain their own genome indicating that they play additionally an eminent role in processes beyond bioenergetics. Despite intense efforts in identifying and characterizing mitochondrial protein complexes by structural biology and proteomics techniques, many PPIs have remained elusive. Several of these (membrane embedded) PPIs are less stable in vitro hampering their characterization by most contemporary methods in structural biology. Particularly in these cases, cross-linking mass spectrometry (XL-MS) has proven valuable for the in-depth characterization of mitochondrial protein complexes in situ. Here, we highlight experimental strategies for the analysis of proteome-wide PPIs in mitochondria using XL-MS. We showcase the ability of in situ XL-MS as a tool to map suborganelle interactions and topologies and aid in refining structural models of protein complexes. We describe some of the most recent technological advances in XL-MS that may benefit the in situ characterization of PPIs even further, especially when combined with electron microscopy and structural modeling.

Intrinsic and extrinsic determinants of conditional localization of Mms6 to magnetosome organelles in Magnetospirillum magneticum AMB-1.

Magnetotactic bacteria are a diverse group of microbes that use magnetic particles housed within intracellular lipid-bounded magnetosome organelles to guide navigation along geomagnetic fields. The development of magnetosomes and their magnetic crystals in Magnetospirillum magneticum AMB-1 requires the coordinated action of numerous proteins. Most proteins are thought to localize to magnetosomes during the initial stages of organelle biogenesis, regardless of environmental conditions. However, the magnetite-shaping protein Mms6 is only found in magnetosomes that contain magnetic particles, suggesting that it might conditionally localize after the formation of magnetosome membranes. The mechanisms for this unusual mode of localization to magnetosomes are unclear. Here, using pulse-chase labeling, we show that Mms6 translated under non-biomineralization conditions translocates to pre-formed magnetosomes when cells are shifted to biomineralizing conditions. Genes essential for magnetite production, namely mamE, mamM, and mamO, are necessary for Mms6 localization, whereas mamN inhibits Mms6 localization. MamD localization was also investigated and found to be controlled by similar cellular factors. The membrane localization of Mms6 is dependent on a glycine-leucine repeat region, while the N-terminal domain of Mms6 is necessary for retention in the cytosol and impacts conditional localization to magnetosomes. The N-terminal domain is also sufficient to impart conditional magnetosome localization to MmsF, altering its native constitutive magnetosome localization. Our work illuminates an alternative mode of protein localization to magnetosomes in which Mms6 and MamD are excluded from magnetosomes by MamN until biomineralization initiates, whereupon they translocate into magnetosome membranes to control the development of growing magnetite crystals.IMPORTANCEMagnetotactic bacteria (MTB) are a diverse group of bacteria that form magnetic nanoparticles surrounded by membranous organelles. MTB are widespread and serve as a model for bacterial organelle formation and biomineralization. Magnetosomes require a specific cohort of proteins to enable magnetite formation, but how those proteins are localized to magnetosome membranes is unclear. Here, we investigate protein localization using pulse-chase microscopy and find a system of protein coordination dependent on biomineralization-permissible conditions. In addition, our findings highlight a protein domain that alters the localization behavior of magnetosome proteins. Utilization of this protein domain may provide a synthetic route for conditional functionalization of magnetosomes for biotechnological applications.

SIRT1 regulates the localization and stability of telomerase protein by direct interaction.

Telomerase reverse transcriptase (TERT) not only upholds telomeric equilibrium but also plays a pivotal role in multiple non-canonical cellular mechanisms, particularly in the context of aging, cancer, and genomic stability. Though depletion of SIRT1 in mouse embryonic fibroblasts has demonstrated telomere shortening, the impact of SIRT1 on enabling TERT to regulate telomeric homeostasis remains enigmatic. Here, we reveal that SIRT1 directly interacts with TERT, and promotes the nuclear localization and stability of TERT. Reverse transcriptase (RT) domain of TERT and N-terminus of SIRT1 mainly participated in their direct interaction. TERT, concomitantly expressed with intact SIRT1, exhibits nuclear localization, whereas TERT co-expressed with N-terminal-deleted SIRT1 remains in the cytosol. Furthermore, overexpression of SIRT1 enhances the nuclear localization and protein stability of TERT, akin to overexpression of deacetylase-inactive SIRT1, whereas N-terminal-deleted SIRT1 has no effect on TERT. These findings suggest a novel regulatory role of SIRT1 for TERT through direct interaction. This interaction provides new insights into the fields of aging, cancer, and genome stability governed by TERT and SIRT1.

Protein research in millets: current status and way forward.

MAIN CONCLUSION: Millets' protein studies are lagging behind those of major cereals. Current status and future insights into the investigation of millet proteins are discussed. Millets are important small-seeded cereals majorly grown and consumed by people in Asia and Africa and are considered crops of future food security. Although millets possess excellent climate resilience and nutrient supplementation properties, their research advancements have been lagging behind major cereals. Although considerable genomic resources have been developed in recent years, research on millet proteins and proteomes is currently limited, highlighting a need for further investigation in this area. This review provides the current status of protein research in millets and provides insights to understand protein responses for climate resilience and nutrient supplementation in millets. The reference proteome data is available for sorghum, foxtail millet, and proso millet to date; other millets, such as pearl millet, finger millet, barnyard millet, kodo millet, tef, and browntop millet, do not have any reference proteome data. Many studies were reported on stress-responsive protein identification in foxtail millet, with most studies on the identification of proteins under drought-stress conditions. Pearl millet has a few reports on protein identification under drought and saline stress. Finger millet is the only other millet to have a report on stress-responsive (drought) protein identification in the leaf. For protein localization studies, foxtail millet has a few reports. Sorghum has the highest number of 40 experimentally proven crystal structures, and other millets have fewer or no experimentally proven structures. Further proteomics studies will help dissect the specific proteins involved in climate resilience and nutrient supplementation and aid in breeding better crops to conserve food security.

Expression of prolyl hydroxylase domains, the upstream regulators of HIF, in the brain of the anoxia-tolerant crucian carp during anoxia-reoxygenation.

The hypoxia-inducible factor (HIF) is considered key in the transcriptional response to low oxygen. Yet, the role of HIF in the absence of oxygen (anoxia) and in preparation for reoxygenation remains unclear. Recent studies suggest that mounting a HIF response may be counterproductive for anoxia survival. We here studied one of the champions of anoxia survival, the crucian carp (Carassius carassius), and hypothesized that expression of prolyl hydroxylase domains (PHDs; the upstream regulators of HIF) are upregulated to circumvent an energy-costly activation of HIF in anoxia and to prepare for reoxygenation. We measured whole brain mRNA and protein levels of the three isoforms PHD1, PHD2, and PHD3, coded for by multiple paralogs of the genes egln2, egln1, and egln3, using quantitative PCR and Western blotting in the brain of crucian carps exposed to 5 days normoxia or anoxia, and 5 days anoxia followed by 3 or 24 h of reoxygenation. The mRNA levels of most egln paralogs were increased in anoxia and upon reoxygenation, with egln3 showing the largest increase in mRNA level (up to 17-fold) and highest relative mRNA abundance (up to 75% of expressed egln). The protein level of all PHDs was maintained in anoxia and increased upon reoxygenation. We then explored PHD distribution in different brain regions and found PHD immunoreactivity to be associated with axonal branches and showing region-specific changes during anoxia-reoxygenation. Our results support an overall upregulation of egln under prolonged anoxia and PHDs upon reoxygenation in crucian carp, likely aimed at suppressing HIF responses, although regional differences are apparent in such a complex organ as the brain.NEW & NOTEWORTHY We report a profound upregulation of most egln paralog mRNA levels in anoxia and upon reoxygenation, with egln3ii showing the largest, a 17-fold increase, and highest relative mRNA abundance. The relative abundance of prolyl hydroxylase domain (PHD) proteins was maintained during anoxia and increased at reoxygenation. PHD immunoreactivity was localized to axonal branches with region-specific changes during anoxia-reoxygenation. These dynamic and regional changes in crucian carp, champion of anoxia tolerance, are most likely adaptive and call for further mechanistic studies.

A chemical magnet: Approaches to guide precise protein localization.

Small molecules that chemically induce proximity between two proteins have been widely used to precisely modulate protein levels, stability, and activity. Recently, several studies developed novel strategies that employ heterobifunctional molecules that co-opt shuttling proteins to control the spatial localization of a target protein, unlocking new potential within this domain. Together, these studies lay the groundwork for novel targeted protein relocalization modalities that can rewire the protein circuitry and interactome to influence biological outcomes.

Tyrosine phosphorylation-dependent localization of TmaR that controls activity of a major bacterial sugar regulator by polar sequestration.

The poles of Escherichia coli cells are emerging as hubs for major sensory systems, but the polar determinants that allocate their components to the pole are largely unknown. Here, we describe the discovery of a previously unannotated protein, TmaR, which localizes to the E. coli cell pole when phosphorylated on a tyrosine residue. TmaR is shown here to control the subcellular localization and activity of the general PTS protein Enzyme I (EI) by binding and polar sequestration of EI, thus regulating sugar uptake and metabolism. Depletion or overexpression of TmaR results in EI release from the pole or enhanced recruitment to the pole, which leads to increasing or decreasing the rate of sugar consumption, respectively. Notably, phosphorylation of TmaR is required to release EI and enable its activity. Like TmaR, the ability of EI to be recruited to the pole depends on phosphorylation of one of its tyrosines. In addition to hyperactivity in sugar consumption, the absence of TmaR also leads to detrimental effects on the ability of cells to survive in mild acidic conditions. Our results suggest that this survival defect, which is sugar- and EI-dependent, reflects the difficulty of cells lacking TmaR to enter stationary phase. Our study identifies TmaR as the first, to our knowledge, E. coli protein reported to localize in a tyrosine-dependent manner and to control the activity of other proteins by their polar sequestration and release.

Diverse roles of TssA-like proteins in the assembly of bacterial type VI secretion systems.

Protein translocation by the bacterial type VI secretion system (T6SS) is driven by a rapid contraction of a sheath assembled around a tube with associated effectors. Here, we show that TssA-like or TagA-like proteins with a conserved N-terminal domain and varying C-terminal domains can be grouped into at least three distinct classes based on their role in sheath assembly. The proteins of the first class increase speed and frequency of sheath assembly and form a stable dodecamer at the distal end of a polymerizing sheath. The proteins of the second class localize to the cell membrane and block sheath polymerization upon extension across the cell. This prevents excessive sheath polymerization and bending, which may result in sheath destabilization and detachment from its membrane anchor and thus result in failed secretion. The third class of these proteins localizes to the baseplate and is required for initiation of sheath assembly. Our work shows that while various proteins share a conserved N-terminal domain, their roles in T6SS biogenesis are fundamentally different.

Phosphorylation of the receptor protein Pex5p modulates import of proteins into peroxisomes.

Peroxisomes are organelles with vital functions in metabolism and their dysfunction is associated with human diseases. To fulfill their multiple roles, peroxisomes import nuclear-encoded matrix proteins, most carrying a peroxisomal targeting signal (PTS) 1. The receptor Pex5p recruits PTS1-proteins for import into peroxisomes; whether and how this process is posttranslationally regulated is unknown. Here, we identify 22 phosphorylation sites of Pex5p. Yeast cells expressing phospho-mimicking Pex5p-S507/523D (Pex5p2D) show decreased import of GFP with a PTS1. We show that the binding affinity between a PTS1-protein and Pex5p2D is reduced. An in vivo analysis of the effect of the phospho-mimicking mutant on PTS1-proteins revealed that import of most, but not all, cargos is affected. The physiological effect of the phosphomimetic mutations correlates with the binding affinity of the corresponding extended PTS1-sequences. Thus, we report a novel Pex5p phosphorylation-dependent mechanism for regulating PTS1-protein import into peroxisomes. In a broader view, this suggests that posttranslational modifications can function in fine-tuning the peroxisomal protein composition and, thus, cellular metabolism.

In Silico Prediction of MYO1C-Rhodopsin Interactions and Its Significance in Protein Localization and Visual Function.

Rods and cones are photoreceptor neurons in the retina that are required for visual sensation in vertebrates, where proper protein localization and compartmentalization are critical for phototransduction and visual function. In human retinal diseases, improper protein transport to the outer segment (OS) or mislocalization of proteins to the inner segment (IS) could lead to impaired visual responses and photoreceptor cell degeneration, causing a loss of visual function. We showed involvement of an unconventional motor protein, MYO1C, in the proper localization of rhodopsin to the OS, where loss of MYO1C in a mammalian model caused mislocalization of rhodopsin to IS and cell bodies, leading to progressively severe retinal phenotypes. In this study, using modeling and docking analysis, we aimed to identify the protein-protein interaction sites between MYO1C and Rhodopsin to establish a hypothesis that a physical interaction between these proteins is necessary for the proper trafficking of rhodopsin and visual function.

A DNA polymerization-independent role for mitochondrial DNA polymerase I-like protein C in African trypanosomes.

Mitochondrial DNA of Trypanosoma brucei and related parasites is a catenated network containing thousands of minicircles and tens of maxicircles, called kinetoplast DNA (kDNA). Replication of a single nucleoid requires at least three DNA polymerase I-like proteins (i.e. POLIB, POLIC and POLID), each showing discrete localizations near the kDNA during S phase. POLIB and POLID have roles in minicircle replication but the specific role of POLIC in kDNA maintenance is less clear. Here, we use an RNA interference (RNAi)-complementation system to dissect the functions of two distinct POLIC regions, i.e. the conserved family A DNA polymerase (POLA) domain and the uncharacterized N-terminal region (UCR). While RNAi complementation with wild-type POLIC restored kDNA content and cell cycle localization of kDNA, active site point mutations in the POLA domain impaired minicircle replication similar to that of POLIB and POLID depletions. Complementation with POLA domain alone abolished the formation of POLIC foci and partially rescued the RNAi phenotype. Furthermore, we provide evidence that the UCR is crucial in cell cycle-dependent protein localization and facilitates proper distribution of progeny networks. This is the first report of a DNA polymerase that impacts on mitochondrial nucleoid distribution.This article has an associated First Person interview with the first author of the paper.

All-in-one: a robust fluorescent fusion protein vector toolbox for protein localization and BiFC analyses in plants.

Fluorescent tagging protein localization (FTPL) and bimolecular fluorescence complementation (BiFC) are popular tools for in vivo analyses of the subcellular localizations of proteins and protein-protein interactions in plant cells. The efficiency of fluorescent fusion protein (FFP) expression analyses is typically impaired when the FFP genes are co-transformed on separate plasmids compared to when all are cloned and transformed in a single vector. Functional genomics applications using FFPs such as a gene family studies also often require the generation of multiple plasmids. Here, to address these needs, we developed an efficient, modular all-in-one (Aio) FFP (AioFFP) vector toolbox, including a set of fluorescently labelled organelle markers, FTPL and BiFC plasmids and associated binary vectors. This toolbox uses Gibson assembly (GA) and incorporates multiple unique nucleotide sequences (UNSs) to facilitate efficient gene cloning. In brief, this system enables convenient cloning of a target gene into various FFP vectors or the insertion of two or more target genes into the same FFP vector in a single-tube GA reaction. This system also enables integration of organelle marker genes or fluorescently fused target gene expression units into a single transient expression plasmid or binary vector. We validated the AioFFP system by testing genes encoding proteins known to be functional in FTPL and BiFC assays. In addition, we performed a high-throughput assessment of the accurate subcellular localizations of an uncharacterized rice CBSX protein subfamily. This modular UNS-guided GA-mediated AioFFP vector toolkit is cost-effective, easy to use and will promote functional genomics research in plants.