Reconstitution of phosphatidylserine transport from chemically defined donor membranes to phosphatidylserine decarboxylase 2 implicates specific lipid domains in the process

Phosphatidylserine (PtdSer) is transported from its site of synthesis in the endoplasmic reticulum to the locus of PtdSer decarboxylase 2 (Psd2p) in the Golgi/vacuole and decarboxylated to form phosphatidylethanolamine. Recent biochemical and genetic evidence has implicated the C2 domain of Psd2p and a membrane-bound form of the phosphatidylinositol binding/transfer protein, PstB2p, as essential for this transport process. We devised a reconstituted system in which chemically defined donor membranes function to transfer PtdSer to the biological acceptor membranes containing Psd2p. The transfer of PtdSer is poor when the donor membranes have a high degree of curvature but markedly enhanced when the membranes are relatively planar (> or =400-nm diameter). PtdSer transfer is also dependent upon both the bulk and the surface concentrations of the lipid, with pure PtdSer vesicles acting as the most efficient donors at all concentrations. The lipid transfer from donor membranes containing either 100% PtdSer or 50% PtdSer at a fixed concentration (e.g. 250 microM PtdSer) differs by a factor of 20. Surface dilution of PtdSer by choline, ethanolamine, glycerol, and inositol phospholipids markedly inhibits PtdSer transfer, whereas phosphatidic acid (PtdOH) stimulates the transfer. Most importantly, the transfer of PtdSer from liposomes to Psd2p fails to occur in acceptor membranes from strains lacking PstB2p or the C2 domain of Psd2p. These data support a model for PtdSer transport from planar domains highly enriched in PtdSer or in PtdSer plus PtdOH.     

New perspectives on the regulation of intermembrane glycerophospholipid traffic

In eukaryotes, phosphatidylserine (PtdSer) can serve as a precursor of phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho), which are the major cellular phospholipids. PtdSer synthesis originates in the endoplasmic reticulum (ER) and its subdomain named the mitochondria-associated membrane (MAM). PtdSer is transported to the mitochondria in mammalian cells and yeast, and decarboxylated by PtdSer decarboxylase 1 (Psd1p) to form PtdEtn. A second decarboxylase, Psd2p, is also found in yeast in the Golgi-vacuole. PtdEtn produced by Psd1p and Psd2p can be transported to the ER, where it is methylated to form PtdCho. Organelle-specific metabolism of the aminoglycerophospholipids is a powerful tool for experimentally following lipid traffic that is now enabling identification of new proteins involved in the regulation of this process. Genetic and biochemical experiments demonstrate that transport of PtdSer between the MAM and mitochondria is regulated by protein ubiquitination, which affects events at both membranes. Similar analyses of PtdSer transport to the locus of Psd2p now indicate that a membrane-bound phosphatidylinositol transfer protein and the C2 domain of Psd2p are both required on the acceptor membrane for efficient transport of PtdSer. Collectively, these recent findings indicate that novel multiprotein assemblies on both donor and acceptor membranes participate in interorganelle phospholipid transport.     

Phosphatidylserine transport to the mitochondria is regulated by ubiquitination

Mitochondrial membrane biogenesis requires the interorganelle transport of phospholipids. Phosphatidylserine (PtdSer) synthesized in the endoplasmic reticulum and related membranes (mitochondria-associated membrane (MAM)) is transported to the mitochondria by unknown gene products and decarboxylated to form phosphatidylethanolamine at the inner membrane by PtdSer decarboxylase 1 (Psd1p). We have designed a screen for strains defective in PtdSer transport (pstA mutants) between the endoplasmic reticulum and Psd1p that relies on isolating ethanolamine auxotrophs in suitable (psd2Delta) genetic backgrounds. Following chemical mutagenesis, we isolated an ethanolamine auxotroph that we designate pstA1-1. Using in vivo and in vitro phospholipid synthesis/transport measurements, we demonstrate that the pstA1-1 mutant is defective in PtdSer transport between the MAM and mitochondria. The gene that complements the growth defect and PtdSer transport defect of the pstA1-1 mutant is MET30, which encodes a substrate recognition subunit of the SCF (suppressor of kinetochore protein 1, cullin, F-box) ubiquitin ligase complex. Reconstitution of different permutations of MAM and mitochondria from wild type and pstA1-1 strains demonstrates that the MET30 gene product affects both organelles. These data provide compelling evidence that interorganelle PtdSer traffic is regulated by ubiquitination.     

Transport of Phosphatidylserine from the Endoplasmic Reticulum to the Site of Phosphatidylserine Decarboxylase2 in Yeast

Over the past two decades, most of the genes specifying lipid synthesis and metabolism in yeast have been identified and characterized. Several of these biosynthetic genes and their encoded enzymes have provided valuable tools for the genetic and biochemical dissection of interorganelle lipid transport processes in yeast. One such pathway involves the synthesis of phosphatidylserine (PtdSer) in the endoplasmic reticulum (ER), and its non‐vesicular transport to the site of phosphatidylserine decarboxylase2 (Psd2p) in membranes of the Golgi and endosomal sorting system. In this review, we summarize the identification and characterization of the yeast phosphatidylserine decarboxylases, and examine their role in studies of the transport‐dependent pathways of de novo synthesis of phosphatidylethanolamine (PtdEtn). The emerging picture of the Psd2p‐specific transport pathway is one in which the enzyme and its non‐catalytic N‐terminal domains act as a hub to nucleate the assembly of a multiprotein complex, which facilitates PtdSer transport at membrane contact sites between the ER and Golgi/endosome membranes. After transport to the catalytic site of Psd2p, PtdSer is decarboxylated to form PtdEtn, which is disseminated throughout the cell to support the structural and functional needs of multiple membranes.      

The C2 domain of phosphatidylserine decarboxylase 2 is not required for catalysis but is essential for in vivo function

Phosphatidylserine decarboxylase 2 (Psd2p) is currently being used to study lipid trafficking processes in intact and permeabilized yeast cells. The Psd2p contains a C2 homology domain and a putative Golgi retention/localization (GR) domain. C2 domains play important functions in membrane binding and docking reactions involving phospholipids and proteins. We constructed a C2 domain deletion variant (C2Delta) and a GR deletion variant (GRDelta) of Psd2p and examined their effects on in vivo function and catalysis. Immunoblotting confirmed that the predicted immature and mature forms of Psd2(C2Delta)p, Psd2(GRDelta)p, and wild type Psd2p were produced in vivo and that the proteins localized normally. Enzymology revealed that the Psd2(C2Delta)p and Psd2(GRDelta)p were catalytically active and could readily be expressed at levels 10-fold higher than endogenous Psd2p. Both Psd2p and Psd2(GRDelta)p expression complemented the growth defect of psd1Deltapsd2Delta strains and resulted in normal aminoglycerophospholipid metabolism. In contrast, the Psd2(C2Delta)p failed to complement psd1Deltapsd2Delta strains, and [(3)H]serine labeling revealed a severe defect in the formation of PtdEtn in both intact and permeabilized cells, indicative of disruption of lipid trafficking. These findings identify an essential, non-catalytic function of the C2 domain of Psd2p and raise the possibility that it plays a direct role in membrane docking and/or PtdSer transport to the enzyme.     

Characterization of phosphatidylserine transport to the locus of phosphatidylserine decarboxylase 2 in permeabilized yeast

In yeast, nascent phosphatidylserine (PtdSer) can be transported to the mitochondria and Golgi/vacuole for decarboxylation to synthesize phosphatidylethanolamine (PtdEtn). In strains with a psd1Delta allele for the mitochondrial PtdSer decarboxylase, the conversion of nascent PtdSer to PtdEtn can serve as an indicator of lipid transport to the locus of PtdSer decarboxylase 2 (Psd2p) in the Golgi/vacuole. We have followed the metabolism of [(3)H]serine into PtdSer and PtdEtn to study lipid transport in permeabilized psd1Delta yeast. The permeabilized cells synthesize (3)H-PtdSer and, after a 20-min lag, decarboxylate it to form [(3)H]PtdEtn. Formation of [(3)H]PtdEtn is linear between 20 and 100 min of incubation and does not require ongoing PtdSer synthesis. PtdSer transport can be resolved into a two-component system using washed, permeabilized psd1Delta cells as donors and membranes isolated by ultracentrifugation as acceptors. With this system, the transport-dependent decarboxylation of nascent PtdSer is dependent upon the concentration of acceptor membranes, requires Mn(2+) but not nucleotides, and is inhibited by EDTA. High speed membranes isolated from a previously identified PtdSer transport mutant, pstB2, contain normal Psd2p activity but fail to reconstitute PtdSer transport and decarboxylation. Reconstitution with permutations of wild type and pstB2Delta donors and acceptors identifies the site of the mutant defect as the acceptor side of the transport reaction.     

Contribution of different biosynthetic pathways to species selectivity of aminoglycerophospholipids assembled into mitochondrial membranes of the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, three pathways lead to the formation of cellular phosphatidylethanolamine (PtdEtn), namely the mitochondrial conversion of phosphatidylserine (PtdSer) to PtdEtn catalyzed by phosphatidylserine decarboxylase 1 (Psd1p), the equivalent reaction catalyzed by phosphatidylserine decarboxylase 2 (Psd2p) in the Golgi, and the CDP-ethanolamine branch of the so-called Kennedy pathway which is located to the microsomal fraction. To investigate the contributions of these three pathways to the cellular pattern of PtdEtn species (fatty acid composition) we subjected lipids of wild-type and yeast mutant strains with distinct defects in the respective pathways to mass spectrometric analysis. We also analyzed species of PtdSer and phosphatidylcholine (PtdCho) of these strains because formation of the three aminoglycerophospholipids is linked through their biosynthetic route. We demonstrate that all three pathways involved in PtdEtn synthesis exhibit a preference for the formation of C34:2 and C32:2 species resulting in a high degree of unsaturation in total cellular PtdEtn. In PtdSer, the ratio of unsaturated to saturated fatty acids is much lower than in PtdEtn, suggesting a high species selectivity of PtdSer decarboxylases. Finally, PtdCho is characterized by its higher ratio of C16 to C18 fatty acids compared to PtdSer and PtdEtn. In contrast to biosynthetic steps, import of all three aminoglycerophospholipids into mitochondria of wild-type and mutant cells is not highly specific with respect to species transported. Thus, the species pattern of aminoglycerophospholipids in mitochondria is mainly the result of enzyme specificities, but not of translocation processes involved. Our results support a model that suggests equilibrium transport of aminoglycerophospholipids between mitochondria and microsomes based on membrane contact between the two compartments.     

Contribution of different pathways to the supply of phosphatidylethanolamine and phosphatidylcholine to mitochondrial membranes of the yeast Saccharomyces cerevisiae

In the yeast, three biosynthetic pathways lead to the formation of phosphatidylethanolamine (PtdEtn): (i) decarboxylation of phosphatidylserine (PtdSer) by phosphatidylserine decarboxylase 1 (Psd1p) in mitochondria; (ii) decarboxylation of PtdSer by Psd2p in a Golgi/vacuolar compartment; and (iii) the CDP-ethanolamine (CDP-Etn) branch of the Kennedy pathway. The major phospholipid of the yeast, phosphatidylcholine (PtdCho), is formed either by methylation of PtdEtn or via the CDP-choline branch of the Kennedy pathway. To study the contribution of these pathways to the supply of PtdEtn and PtdCho to mitochondrial membranes, labeling experiments in vivo with [(3)H]serine and [(14)C]ethanolamine, or with [(3)H]serine and [(14)C]choline, respectively, and subsequent cell fractionation were performed with psd1Delta and psd2Delta mutants. As shown by comparison of the labeling patterns of the different strains, the major source of cellular and mitochondrial PtdEtn is Psd1p. PtdEtn formed by Psd2p or the CDP-Etn pathway, however, can be imported into mitochondria, although with moderate efficiency. In contrast to mitochondria, microsomal PtdEtn is mainly derived from the CDP-Etn pathway. PtdEtn formed by Psd2p is the preferred substrate for PtdCho synthesis. PtdCho derived from the different pathways appears to be supplied to subcellular membranes from a single PtdCho pool. Thus, the different pathways of PtdEtn biosynthesis play different roles in the assembly of PtdEtn into cellular membranes.     

Processing and Topology of the Yeast Mitochondrial Phosphatidylserine Decarboxylase 1

The inner mitochondrial membrane plays a crucial role in cellular lipid homeostasis through biosynthesis of the non-bilayer-forming lipids phosphatidylethanolamine and cardiolipin. In the yeast Saccharomyces cerevisiae, the majority of cellular phosphatidylethanolamine is synthesized by the mitochondrial phosphatidylserine decarboxylase 1 (Psd1). The biogenesis of Psd1 involves several processing steps. It was speculated that the Psd1 precursor is sorted into the inner membrane and is subsequently released into the intermembrane space by proteolytic removal of a hydrophobic sorting signal. However, components involved in the maturation of the Psd1 precursor have not been identified. We show that processing of Psd1 involves the action of the mitochondrial processing peptidase and Oct1 and an autocatalytic cleavage at a highly conserved LGST motif yielding the α- and β-subunit of the enzyme. The Psd1 β-subunit (Psd1β) forms the membrane anchor, which binds the intermembrane space-localized α-subunit (Psd1α). Deletion of a transmembrane segment in the β-subunit results in mislocalization of Psd1 and reduced enzymatic activity. Surprisingly, autocatalytic cleavage does not depend on proper localization to the inner mitochondrial membrane. In summary, membrane integration of Psd1 is crucial for its functionality and for maintenance of mitochondrial lipid homeostasis.     

A new gene involved in the transport-dependent metabolism of phosphatidylserine, PSTB2/PDR17, shares sequence similarity with the gene encoding the phosphatidylinositol/phosphatidylcholine transfer protein, SEC14

A new yeast strain, designated pstB2, that is defective in the conversion of nascent phosphatidylserine (PtdSer) to phosphatidylethanolamine (PtdEtn) by PtdSer decarboxylase 2, has been isolated. The pstB2 strain requires ethanolamine for growth. Incubation of cells with [(3)H]serine followed by analysis of the aminoglycerophospholipids demonstrates a 50% increase in the labeling of PtdSer and a 72% decrease in PtdEtn formation in the mutant relative to the parental strain. The PSTB2 gene was isolated by complementation, and it restores ethanolamine prototrophy and corrects the defective lipid metabolism of the pstB2 strain. The PSTB2 gene is allelic to the pleiotropic drug resistance gene, PDR17, and is homologous to SEC14, which encodes a phosphatidylinositol/phosphatidylcholine transfer protein. The protein, PstB2p, displays phosphatidylinositol but not PtdSer transfer activity, and its overexpression causes suppression of sec14 mutants. However, overexpression of the SEC14 gene fails to suppress the conditional lethality of pstB2 strains. The transport-dependent metabolism of PtdSer to PtdEtn occurs in permeabilized wild type yeast but is dramatically reduced in permeabilized pstB2 strains. Fractionation of permeabilized cells demonstrates that the pstB2 strain accumulates nascent PtdSer in the Golgi apparatus and a novel light membrane fraction, consistent with a defect in lipid transport processes that control substrate access to PtdSer decarboxylase 2.     

Ist2 recruits the lipid transporters Osh6/7 to ER–PM contacts to maintain phospholipid metabolism

ER-plasma membrane (PM) contacts are proposed to be held together by distinct families of tethering proteins, which in yeast include the VAP homologues Scs2/22, the extended-synaptotagmin homologues Tcb1/2/3, and the TMEM16 homologue Ist2. It is unclear whether these tethers act redundantly or whether individual tethers have specific functions at contacts. Here, we show that Ist2 directly recruits the phosphatidylserine (PS) transport proteins and ORP family members Osh6 and Osh7 to ER–PM contacts through a binding site located in Ist2’s disordered C-terminal tethering region. This interaction is required for phosphatidylethanolamine (PE) production by the PS decarboxylase Psd2, whereby PS transported from the ER to the PM by Osh6/7 is endocytosed to the site of Psd2 in endosomes/Golgi/vacuoles. This role for Ist2 and Osh6/7 in nonvesicular PS transport is specific, as other tethers/transport proteins do not compensate. Thus, we identify a molecular link between the ORP and TMEM16 families and a role for endocytosis of PS in PE synthesis.     

Phosphatidylserine Decarboxylase 1 Autocatalysis and Function Does Not Require a Mitochondrial-specific Factor

Phosphatidylethanolamine (PE) is a major cellular phospholipid that can be made by four separate pathways, one of which resides in the mitochondrion. The mitochondrial enzyme that generates PE is phosphatidylserine decarboxylase 1 (Psd1p). The pool of PE produced by Psd1p, which cannot be compensated for by the other cellular PE metabolic pathways, is important for numerous mitochondrial functions, including oxidative phosphorylation and mitochondrial dynamics and morphology, and is essential for murine development. To become catalytically active, Psd1p undergoes an autocatalytic processing step involving a conserved LGST motif that separates the enzyme into α and β subunits that remain non-covalently attached and are anchored to the inner membrane by virtue of the membrane-embedded β subunit. It was speculated that Psd1p autocatalysis requires a mitochondrial-specific factor and that for Psd1p to function in vivo, it had to be embedded with the correct topology in the mitochondrial inner membrane. However, the identity of the mitochondrial factor required for Psd1p autocatalysis has not been identified. With the goal of defining molecular requirements for Psd1p autocatalysis, we demonstrate that: 1) despite the conservation of the LGST motif from bacteria to humans, only the serine residue is absolutely required for Psd1p autocatalysis and function; 2) yeast Psd1p does not require its substrate phosphatidylserine for autocatalysis; and 3) contrary to a prior report, yeast Psd1p autocatalysis does not require mitochondrial-specific phospholipids, proteins, or co-factors, because Psd1p re-directed to the secretory pathway undergoes autocatalysis normally and is fully functional in vivo.     

Identification and characterization of the mitochondrial membrane sorting signals in phosphatidylserine decarboxylase 1 from Saccharomyces cerevisiae

Phosphatidylserine decarboxylase 1 (Psd1p) catalyzes the formation of the majority of phosphatidylethanolamine (PE) in the yeast Saccharomyces cerevisiae. Psd1p is localized to mitochondria, anchored to the inner mitochondrial membrane (IMM) through membrane spanning domains and oriented towards the mitochondrial intermembrane space. We found that Psd1p harbors at least two inner membrane-associated domains, which we named IM1 and IM2. IM1 is important for proper orientation of Psd1p within the IMM (Horvath et al., J. Biol. Chem. 287 (2012) 36744–55), whereas it remained unclear whether IM2 is important for membrane-association of Psd1p. To discover the role of IM2 in Psd1p import, processing and assembly into the mitochondria, we constructed Psd1p variants with deletions in IM2. Removal of the complete IM2 led to an altered topology of the protein with the soluble domain exposed to the matrix and to decreased enzyme activity. Psd1p variants lacking portions of the N-terminal moiety of IM2 were inserted into IMM with an altered topology. Psd1p variants with deletions of C-terminal portions of IM2 accumulated at the outer mitochondrial membrane and lost their enzyme activity. In conclusion we showed that IM2 is essential for full enzymatic activity, maturation and correct integration of yeast Psd1p into the inner mitochondrial membrane.     

A Conserved Interaction between the SDI Domain of Bre2 and the Dpy-30 Domain of Sdc1 Is Required for Histone Methylation and Gene Expression

In Saccharomyces cerevisiae, lysine 4 on histone H3 (H3K4) is methylated by the Set1 complex (Set1C or COMPASS). Besides the catalytic Set1 subunit, several proteins that form the Set1C (Swd1, Swd2, Swd3, Spp1, Bre2, and Sdc1) are also needed to mediate proper H3K4 methylation. Until this study, it has been unclear how individual Set1C members interact and how this interaction may impact histone methylation and gene expression. In this study, Bre2 and Sdc1 are shown to directly interact, and it is shown that the association of this heteromeric complex is needed for proper H3K4 methylation and gene expression to occur. Interestingly, mutational and biochemical analysis identified the C terminus of Bre2 as a critical protein-protein interaction domain that binds to the Dpy-30 domain of Sdc1. Using the human homologs of Bre2 and Sdc1, ASH2L and DPY-30, respectively, we demonstrate that the C terminus of ASH2L also interacts with the Dpy-30 domain of DPY-30, suggesting that this protein-protein interaction is maintained from yeast to humans. Because of the functionally conserved nature of the C terminus of Bre2 and ASH2L, this region was named the SDI (Sdc1 Dpy-30 interaction) domain. Finally, we show that the SDI-Dpy-30 domain interaction is physiologically important for the function of Set1 in vivo, because specific disruption of this interaction prevents Bre2 and Sdc1 association with Set1, resulting in H3K4 methylation defects and decreases in gene expression. Overall, these and other mechanistic studies on how H3K4 methyltransferase complexes function will likely provide insights into how human MLL and SET1-like complexes or overexpression of ASH2L leads to oncogenesis.     

Structure and Function of the PLAA/Ufd3-p97/Cdc48 Complex

PLAA (ortholog of yeast Doa1/Ufd3, also know as human PLAP or phospholipase A2-activating protein) has been implicated in a variety of disparate biological processes that involve the ubiquitin system. It is linked to the maintenance of ubiquitin levels, but the mechanism by which it accomplishes this is unclear. The C-terminal PUL (PLAP, Ufd3p, and Lub1p) domain of PLAA binds p97, an AAA ATPase, which among other functions helps transfer ubiquitinated proteins to the proteasome for degradation. In yeast, loss of Doa1 is suppressed by altering p97/Cdc48 function indicating that physical interaction between PLAA and p97 is functionally important. Although the overall regions of interaction between these proteins are known, the structural basis has been unavailable. We solved the high resolution crystal structure of the p97-PLAA complex showing that the PUL domain forms a 6-mer Armadillo-containing domain. Its N-terminal extension folds back onto the inner curvature forming a deep ridge that is positively charged with residues that are phylogenetically conserved. The C terminus of p97 binds in this ridge, where the side chain of p97-Tyr⁸⁰⁵, implicated in phosphorylation-dependent regulation, is buried. Expressed in doa1Δ null cells, point mutants of the yeast ortholog Doa1 that disrupt this interaction display slightly reduced ubiquitin levels, but unlike doa1Δ null cells, showed only some of the growth phenotypes. These data suggest that the p97-PLAA interaction is important for a subset of PLAA-dependent biological processes and provides a framework to better understand the role of these complex molecules in the ubiquitin system.     

Collagen-binding Microbial Surface Components Recognizing Adhesive Matrix Molecule (MSCRAMM) of Gram-positive Bacteria Inhibit Complement Activation via the Classical Pathway

Members of a family of collagen-binding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) from Gram-positive bacteria are established virulence factors in several infectious diseases models. Here, we report that these adhesins also can bind C1q and act as inhibitors of the classical complement pathway. Molecular analyses of Cna from Staphylococcus aureus suggested that this prototype MSCRAMM bound to the collagenous domain of C1q and interfered with the interactions of C1r with C1q. As a result, C1r₂C1s₂ was displaced from C1q, and the C1 complex was deactivated. This novel function of the Cna-like MSCRAMMs represents a potential immune evasion strategy that could be used by numerous Gram-positive pathogens.     

Structural Mechanism for the Specific Assembly and Activation of the Extracellular Signal Regulated Kinase 5 (ERK5) Module

Mitogen-activated protein kinase (MAPK) activation depends on a linear binding motif found in all MAPK kinases (MKK). In addition, the PB1 (Phox and Bem1) domain of MKK5 is required for extracellular signal regulated kinase 5 (ERK5) activation. We present the crystal structure of ERK5 in complex with an MKK5 construct comprised of the PB1 domain and the linear binding motif. We show that ERK5 has distinct protein-protein interaction surfaces compared with ERK2, which is the closest ERK5 paralog. The two MAPKs have characteristically different physiological functions and their distinct protein-protein interaction surface topography enables them to bind different sets of activators and substrates. Structural and biochemical characterization revealed that the MKK5 PB1 domain cooperates with the MAPK binding linear motif to achieve substrate specific binding, and it also enables co-recruitment of the upstream activating enzyme and the downstream substrate into one signaling competent complex. Studies on present day MAPKs and MKKs hint on the way protein kinase networks may evolve. In particular, they suggest how paralogous enzymes with similar catalytic properties could acquire novel signaling roles by merely changing the way they make physical links to other proteins.     

Zeta Inhibitory Peptide Disrupts Electrostatic Interactions That Maintain Atypical Protein Kinase C in Its Active Conformation on the Scaffold p62

Atypical protein kinase C (aPKC) enzymes signal on protein scaffolds, yet how they are maintained in an active conformation on scaffolds is unclear. A myristoylated peptide based on the autoinhibitory pseudosubstrate fragment of the atypical PKCζ, zeta inhibitory peptide (ZIP), has been extensively used to inhibit aPKC activity; however, we have previously shown that ZIP does not inhibit the catalytic activity of aPKC isozymes in cells (Wu-Zhang, A. X., Schramm, C. L., Nabavi, S., Malinow, R., and Newton, A. C. (2012) J. Biol. Chem. 287, 12879–12885). Here we sought to identify a bona fide target of ZIP and, in so doing, unveiled a novel mechanism by which aPKCs are maintained in an active conformation on a protein scaffold. Specifically, we used protein-protein interaction network analysis, structural modeling, and protein-protein docking to predict that ZIP binds an acidic surface on the Phox and Bem1 (PB1) domain of p62, an interaction validated by peptide array analysis. Using a genetically encoded reporter for PKC activity fused to the p62 scaffold, we show that ZIP inhibits the activity of wild-type aPKC, but not a construct lacking the pseudosubstrate. These data support a model in which the pseudosubstrate of aPKCs is tethered to the acidic surface on p62, locking aPKC in an open, signaling-competent conformation. ZIP competes for binding to the acidic surface, resulting in displacement of the pseudosubstrate of aPKC and re-engagement in the substrate-binding cavity. This study not only identifies a cellular target for ZIP, but also unveils a novel mechanism by which scaffolded aPKC is maintained in an active conformation.     

BRIZ1 and BRIZ2 Proteins Form a Heteromeric E3 Ligase Complex Required for Seed Germination and Post-germination Growth in Arabidopsis thaliana

Ubiquitin pathway E3 ligases are an important component conferring specificity and regulation in ubiquitin attachment to substrate proteins. The Arabidopsis thaliana RING (Really Interesting New Gene) domain-containing proteins BRIZ1 and BRIZ2 are essential for normal seed germination and post-germination growth. Loss of either BRIZ1 (At2g42160) or BRIZ2 (At2g26000) results in a severe phenotype. Heterozygous parents produce progeny that segregate 3:1 for wild-type:growth-arrested seedlings. Homozygous T-DNA insertion lines are recovered for BRIZ1 and BRIZ2 after introduction of a transgene containing the respective coding sequence, demonstrating that disruption of BRIZ1 or BRIZ2 in the T-DNA insertion lines is responsible for the observed phenotype. Both proteins have multiple predicted domains in addition to the RING domain as follows: a BRAP2 (BRCA1-Associated Protein 2), a ZnF UBP (Zinc Finger Ubiquitin Binding protein), and a coiled-coil domain. In vitro, both BRIZ1 and BRIZ2 are active as E3 ligases but only BRIZ2 binds ubiquitin. In vitro synthesized and purified recombinant BRIZ1 and BRIZ2 preferentially form hetero-oligomers rather than homo-oligomers, and the coiled-coil domain is necessary and sufficient for this interaction. BRIZ1 and BRIZ2 co-purify after expression in tobacco leaves, which also requires the coiled-coil domain. BRIZ1 and BRIZ2 coding regions with substitutions in the RING domain are inactive in vitro and, after introduction, fail to complement their respective mutant lines. In our current model, BRIZ1 and BRIZ2 together are required for formation of a functional ubiquitin E3 ligase in vivo, and this complex is required for germination and early seedling growth.