Modularity of membrane-bound charge-translocating protein complexes

Energy transduction is the conversion of one form of energy into another; this makes life possible as we know it. Organisms have developed different systems for acquiring energy and storing it in useable forms: the so-called energy currencies. A universal energy currency is the transmembrane difference of electrochemical potential (Δμ~). This results from the translocation of charges across a membrane, powered by exergonic reactions. Different reactions may be coupled to charge-translocation and, in the majority of cases, these reactions are catalyzed by modular enzymes that always include a transmembrane subunit. The modular arrangement of these enzymes allows for different catalytic and charge-translocating modules to be combined. Thus, a transmembrane charge-translocating module can be associated with different catalytic subunits to form an energy-transducing complex. Likewise, the same catalytic subunit may be combined with a different membrane charge-translocating module. In this work, we analyze the modular arrangement of energy-transducing membrane complexes and discuss their different combinations, focusing on the charge-translocating module.

Native mass spectrometry identifies the HybG chaperone as carrier of the Fe(CN)2CO group during maturation of E. coli [NiFe]-hydrogenase 2

Abstract[NiFe]-hydrogenases activate dihydrogen. Like all [NiFe]-hydrogenases, hydrogenase 2 of Escherichia coli has a bimetallic NiFe(CN)2CO cofactor in its catalytic subunit. Biosynthesis of the Fe(CN)2CO group of the [NiFe]-cofactor occurs on a distinct scaffold complex comprising the HybG and HypD accessory proteins. HybG is a member of the HypC-family of chaperones that confers specificity towards immature hydrogenase catalytic subunits during transfer of the Fe(CN)2CO group. Using native mass spectrometry of an anaerobically isolated HybG–HypD complex we show that HybG carries the Fe(CN)2CO group. Our results also reveal that only HybG, but not HypD, interacts with the apo-form of the catalytic subunit. Finally, HybG was shown to have two distinct, and apparently CO2-related, covalent modifications that depended on the presence of the N-terminal cysteine residue on the protein, possibly representing intermediates during Fe(CN)2CO group biosynthesis. Together, these findings suggest that the HybG chaperone is involved in both biosynthesis and delivery of the Fe(CN)2CO group to its target protein. HybG is thus suggested to shuttle between the assembly complex and the apo-catalytic subunit. This study provides new insights into our understanding of how organometallic cofactor components are assembled on a scaffold complex and transferred to their client proteins.

Substrate recognition determinants of human eIF2α phosphatases

Phosphorylation of the translation initiation factor eIF2α is a rapid and vital cellular defence against many forms of stress. In mammals, the levels of eIF2α phosphorylation are set through the antagonistic action of four protein kinases and two heterodimeric protein phosphatases. The phosphatases are composed of the catalytic subunit PP1 and one of two related non-catalytic subunits, PPP1R15A or PPP1R15B (R15A or R15B). Here, we generated a series of R15 truncation mutants and tested their properties in mammalian cells. We show that substrate recruitment is encoded by an evolutionary conserved region in R15s, R15A 325–554 and R15B 340–639 . G-actin, which has been proposed to confer selectivity to R15 phosphatases, does not bind these regions, indicating that it is not required for substrate binding. Fragments containing the substrate-binding regions but lacking the PP1-binding motif trapped the phospho-substrate and caused accumulation of phosphorylated eIF2α in unstressed cells. Activity assays in cells showed that R15A 325–674 and R15B 340–713 , encompassing the substrate-binding region and the PP1-binding region, exhibit wild-type activity. This work identifies the substrate-binding region in R15s, that functions as a phospho-substrate trapping mutant, thereby defining a key region of R15s for follow up studies.

Regulation of PP2A, PP4, and PP6 holoenzyme assembly by carboxyl-terminal methylation

The family of Phosphoprotein Phosphatases (PPPs) is responsible for most cellular serine and threonine dephosphorylation. PPPs achieve substrate specificity and selectivity by forming multimeric holoenzymes. PPP holoenzyme assembly is tightly controlled, and changes in the cellular repertoire of PPPs are linked to human disease, including cancer and neurodegeneration. For PP2A, PP4, and PP6, holoenzyme formation is in part regulated by carboxyl (C)-terminal methyl-esterification (often referred to as “methylation”). Here, we use mass spectrometry-based proteomics, methylation-ablating mutations, and genome editing to elucidate the role of C-terminal methylation on PP2A, PP4, and PP6 holoenzyme assembly. We find that the catalytic subunits of PP2A, PP4, and PP6 are frequently methylated in cancer cells and that deletion of the C-terminal leucine faithfully recapitulates loss of methylation. We observe that loss of PP2A methylation consistently reduced B55, B56, and B72 regulatory subunit binding in cancer and non-transformed cell lines. However, Striatin subunit binding is only affected in non-transformed cells. For PP4, we find that PP4R1 and PP4R3β bind in a methylation-dependent manner. Intriguingly, loss of methylation does not affect PP6 holoenzymes. Our analyses demonstrate in an unbiased, comprehensive, and isoform-specific manner the crucial regulatory function of endogenous PPP methylation in transformed and non-transformed cell lines.

The Anaphase Promoting Complex/Cyclosome Subunit 11 and Its Role in Organ Size and Plant Development

The anaphase promoting complex/cyclosome (APC/C), a member of the E3 ubiquitin ligase family, plays an important role in recognizing the substrates to be ubiquitylated. Progression of anaphase, and therefore, of the cell cycle, is coordinated through cyclin degradation cycles dependent on proteolysis triggered by APC/C. The APC/C activity depends on the formation of a pocket comprising the catalytic subunits, APC2, APC11, and APC10. Among these, the role of APC11 outside the cell division cycle is poorly understood. Therefore, the goal of this work was to analyze the function of APC11 during plant development by characterizing apc11 knock-down mutant lines. Accordingly, we observed decreased apc11 expression in the mutant lines, followed by a reduction in meristem root size based on the cortical cell length, and an overall size diminishment throughout the development. Additionally, crosses of apc11-1 and amiR-apc11 with plants carrying a WUSCHEL-RELATED HOMEOBOX5 (WOX5) fluorescent marker showed a weakening of the green fluorescent protein-positive cells in the Quiescent Center. Moreover, plants with apc11-1 show a decreased leaf area, together with a decrease in the cell area when the shoot development was observed by kinematics analysis. Finally, we observed a decreased APC/C activity in the root and shoot meristems in crosses of pCYCB1;1:D-box-GUS with apc11-1 plants. Our results indicate that APC11 is important in the early stages of development, mediating meristematic architecture through APC/C activity affecting the overall plant growth.

Regulation of PP2A, PP4, and PP6 holoenzyme assembly by carboxyl-terminal methylation

The family of Phosphoprotein Phosphatases (PPPs) is responsible for most cellular serine and threonine dephosphorylation. PPPs achieve substrate specificity and selectivity by forming multimeric holoenzymes. PPP holoenzyme assembly is tightly controlled, and changes in the cellular repertoire of PPPs are linked to human disease, including cancer and neurodegeneration. For PP2A, PP4, and PP6, holoenzyme formation is in part regulated by carboxyl (C)-terminal methyl-esterification (often referred to as methylation). Here, we use mass spectrometry-based proteomics, methylation-ablating mutations, and genome editing to elucidate the role of C-terminal methylation on PP2A, PP4, and PP6 holoenzyme assembly. We find that the catalytic subunits of PP2A, PP4, and PP6 are frequently methylated in cancer cells and that deletion of the C-terminal leucine faithfully recapitulates loss of methylation. We observe that loss of PP2A methylation consistently reduced B55, B56, and B72 regulatory subunit binding in cancer and non-transformed cell lines. However, Striatin subunit binding is only affected in non-transformed cells. For PP4, we find that PP4R1 and PP4R3β bind in a methylation-dependent manner. Intriguingly, loss of methylation does not affect PP6 holoenzymes. Our analyses demonstrate in an unbiased, comprehensive, and isoform-specific manner the crucial regulatory function of endogenous PPP methylation in transformed and non-transformed cell lines.

Quantitative Genetics of Smoltification Status at the Time of Seawater Transfer in Atlantic Salmon (Salmo Salar)

High mortality during grow out in the sea is a challenge for farmed Atlantic salmon production in Norway and globally, which is partly attributed to suboptimal smolt quality. In this study, two groups of pre-smolts were put on a standard light smoltification regime with alternating 12L:12D per day for 6 weeks (Phase I), followed by 24L:0D per day for 6 weeks (Phase II); one group was 0 + smolt (EXP1) and the other 1 + smolt (EXP2). To monitor the smoltification status of the fish, 100 (EXP1) and 60 (EXP2) fish were randomly sampled per week during Phase II. The following phenotypes for smoltification status were studied: RT-qPCR relative mRNA expression of values of two alpha catalytic subunits of the variants of the Na+K+ATPase (NKA) expressed in the sampled gill tissues of each fish. The first variant, alpha1a with increased expression in freshwater (FW) and the second variant alpha1b with increased expression in seawater variant (SW), as well as their ratio SW/FW. At the optimal time for seawater transfer based on the SW/FW trait, 1,000 (at sixth sampling of EXP1) and 1,500 (at fifth sampling of EXP2) fish were sampled for genetic parameter estimation. The individual variation in FW, SW, and SW/FW was very large at each of the seven samplings indicating a large variation among individuals in the optimum time of transfer to seawater. SW/FW showed significant genetic variation in both 0+ and 1+ smolts, which indicates the possibility for selection for improved synchronization of smoltification status of Atlantic salmon at the time where the largest proportion of the fish is considered to be smolt. However, the genetic correlation between SW/FW of 0+ and 1+ was not significantly different from zero indicating very little shared genetic variation in SW/FW in 0+ and 1+ fish. Smoltification phenotypes showed temporal progression over the smoltification period, and this progression varied between 0+ and 1+ smolt highlighting the importance of correctly timing the major sampling point, and when cohorts are transferred to seawater. This also highlighted the need for further research into noninvasive methods of objectively measuring individual smoltification through time and subsequent smolt survival and growth rate at sea.

STOICHIOMETRY OF THE SODIUM PUMP-PHOSPHOLEMMAN REGULATORY COMPLEX

The sodium-potassium ATPase (NKA) establishes ion gradients that facilitate many physiological processes. In the heart, NKA activity is regulated by its interaction with phospholemman (PLM, FXYD1). Here we used a novel fluorescence lifetime-based assay to investigate the structure, stoichiometry, and affinity of the NKA-PLM regulatory complex. We observed concentration dependent association of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit followed by lower affinity alpha-alpha and alpha-PLM interactions. The data provide the first evidence that the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits in intact cells. Docking and molecular dynamics simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with stoichiometry (alpha-beta-PLM)2.

The peroxisomal exportomer directly inhibits phosphoactivation of the pexophagy receptor Atg36 to suppress pexophagy in yeast

Autophagy receptor (or adaptor) proteins facilitate lysosomal destruction of various organelles in response to cellular stress, including nutrient deprivation. To what extent membrane-resident autophagy receptors also respond to organelle-restricted cues to induce selective autophagy remains poorly understood. We find that latent activation of the yeast pexophagy receptor Atg36 by the casein kinase Hrr25 in rich media is repressed by the ATPase activity of Pex1/6, the catalytic subunits of the exportomer AAA+ transmembrane complex enabling protein import into peroxisomes. Quantitative proteomics of purified Pex3, an obligate Atg36 co-receptor, support a model in which exportomer represses Atg36 without assistance from additional membrane factors. Indeed, we reconstitute inhibition of Atg36 phosphorylation in vitro using soluble Pex1/6 and define an N-terminal unstructured region of Atg36 that enables regulation by binding to Pex1. Our findings uncover a mechanism by which a compartment-specific AAA+ complex mediating organelle biogenesis and protein quality control staves off induction of selective autophagy.