Phosphatidate phosphatase,a key regulator of lipid homeostasis

Yeast Pah1p phosphatidate phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol. PAP plays a crucial role in lipid homeostasis by controlling the relative proportions of its substrate phosphatidate and its product diacylglycerol. The cellular amounts of these lipid intermediates influence the synthesis of triacylglycerol and the pathways by which membrane phospholipids are synthesized. Physiological functions affected by PAP activity include phospholipid synthesis gene expression, nuclear/endoplasmic reticulum membrane growth, lipid droplet formation, and vacuole homeostasis and fusion. Yeast lacking Pah1p PAP activity are acutely sensitive to fatty acid-induced toxicity and exhibit respiratory deficiency. PAP is distinguished in its cellular location, catalytic mechanism, and physiological functions from Dpp1p and Lpp1p lipid phosphate phosphatases that utilize a variety of substrates that include phosphatidate. Phosphorylation/dephosphorylation is a major mechanism by which Pah1p PAP activity is regulated. Pah1p is phosphorylated by cytosolic-associated Pho85p–Pho80p, Cdc28p-cyclin B, and protein kinase A and is dephosphorylated by the endoplasmic reticulum-associated Nem1p–Spo7p phosphatase. The dephosphorylation of Pah1p stimulates PAP activity and facilitates the association with the membrane/phosphatidate allowing for its reaction and triacylglycerol synthesis. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Effect of protein kinase C on endoplasmic reticulum cholesterol.

Plasma membrane cholesterol both regulates and is regulated by effector proteins in the endoplasmic reticulum (ER) through a feedback system that is poorly understood. We now show that ER cholesterol varies over a fivefold range in response to experimental agents that act upon protein kinase C (PKC). Agents that activate Ca(2+)-dependent PKC [phorbol-12-myristate-13-acetate (PMA) and bryostatin 1] increased the level of ER cholesterol; inhibitors such as staurosporine and calphostin C decreased it. Rottlerin, a selective inhibitor of the PKC-delta isoform, also increased ER cholesterol. The esterification of plasma membrane cholesterol was altered by protein kinase C-directed agents in a corresponding fashion. Furthermore, the regulatory effect of plasma membrane cholesterol on the esterification of ER cholesterol was blocked by PKC-directed agents. These findings suggest that multiple protein kinase C isoforms participate in the regulation of ER cholesterol and therefore in cholesterol homeostasis.     

线粒体分裂、融合与细胞凋亡

线粒体是高度动态变化的细胞器,其在细胞内不断分裂、融合并形成网状结构。线粒体的分裂和融合是由多种蛋白质精确调控完成的。Drp1／Dnm1p,Fis1／Fis1p,Caf4p和Mdv1p参与线粒体分裂的调控;Mfn1／2／Fzo1p控制线粒体外膜的融合,而Mgm1p／OPA1则参与线粒体内膜的融合。在细胞凋亡过程中线粒体片段化,网状结构被破坏,线粒体嵴发生重构,抑制这一过程可以部分抑制细胞色素c的释放和细胞凋亡。线粒体形态对于细胞维持正常生理代谢和机体发育起着重要的作用,一旦出现障碍会导致严重的疾病。     

Genetic interactions between KAR7/SEC71, KAR8/JEM1, KAR5, and KAR2 during nuclear fusion in Saccharomyces cerevisiae

During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Delta, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Delta and sec72Delta, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein-protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.     

The Yip1p.Yif1p complex is required for the fusion competence of endoplasmic reticulum-derived vesicles

Here we report that Yip1p and Yif1p, two members of an integral membrane protein complex that bind to the Rab Ypt1p, are required for membrane fusion with the Golgi in vitro. To block fusion, anti-Yip1p or anti-Yif1p antibodies must be added before vesicles bud from the endoplasmic reticulum (ER). These antibodies do not block the packaging of Yip1p, Yif1p, or the soluble NSF attachment protein receptor (SNAREs) into vesicles. We propose that Yip1p and Yif1p perform a critical role in establishing the fusion competence of ER to Golgi vesicles at the time of budding. Consistent with this proposal, we observe that the Yip1p.Yif1p complex binds to the ER to Golgi SNAREs Bos1p and Sec22p, two components of the membrane fusion machinery.     

Protein dislocation from the endoplasmic reticulum--pulling out the suspect

Proteins that fail to fold properly as well as constitutive or regulated short-lived proteins of the endoplasmic reticulum are subjected to proteolysis by cytosolic 26S proteasomes. This process is known as endoplasmic reticulum-associated protein degradation. In order to become accessible to the proteasome of this system substrates must first be retrogradely transported from the endoplasmic reticulum into the cytosol, in a process termed dislocation. This export step seems to be accompanied by polyubiquitination of such molecules. Surprisingly, protein dislocation from the endoplasmic reticulum seems to require at least some components that mediate import into this compartment. However, protein import and export display differences in the mechanism that provides the driving force and ensures directionality. Of special interest is the cytoplasmic Cdc48p/Npl4p/Ufd1p complex, which is required for the degradation of various endoplasmic reticulum-associated protein degradation substrates and seems to function in a step after polyubiquitination but before proteasomal digestion. In this review, we will summarize our knowledge on protein export during endoplasmic reticulum-associated protein degradation and discuss the possible function of certain components involved in this process.     

The hypervariable region of atlastin-1 is a site for intrinsic and extrinsic regulation

Atlastin (ATL) GTPases catalyze homotypic membrane fusion of the peripheral endoplasmic reticulum (ER). GTP-hydrolysis–driven conformational changes and membrane tethering are prerequisites for proper membrane fusion. However, the molecular basis for regulation of these processes is poorly understood. Here we establish intrinsic and extrinsic modes of ATL1 regulation that involve the N-terminal hypervariable region (HVR) of ATLs. Crystal structures of ATL1 and ATL3 exhibit the HVR as a distinct, isoform-specific structural feature. Characterizing the functional role of ATL1’s HVR uncovered its positive effect on membrane tethering and on ATL1’s cellular function. The HVR is post-translationally regulated through phosphorylation-dependent modification. A kinase screen identified candidates that modify the HVR site specifically, corresponding to the modifications on ATL1 detected in cells. This work reveals how the HVR contributes to efficient and potentially regulated activity of ATLs, laying the foundation for the identification of cellular effectors of ATL-mediated membrane processes.     

Tyrosine Phosphorylation Regulates Cell Cycle-dependent Nuclear Localization of Cdc48p

Cdc48p from Saccharomyces cerevisiae and its highly conserved mammalian homologue VCP (valosin-containing protein) are ATPases with essential functions in cell division and homotypic fusion of endoplasmic reticulum vesicles. Both are mainly attached to the endoplasmic reticulum, but relocalize in a cell cycle-dependent manner: Cdc48p enters the nucleus during late G1; VCP aggregates at the centrosome during mitosis. The nuclear import signal sequence of Cdc48p was localized near the amino terminus and its function demonstrated by mutagenesis. The nuclear import is regulated by a cell cycle-dependent phosphorylation of a tyrosine residue near the carboxy terminus. Two-hybrid studies indicate that the phosphorylation results in a conformational change of the protein, exposing the nuclear import signal sequence previously masked by a stretch of acidic residues.     

Properties of a GTP sensitive microdomain in rough microsomes

Stripped rough microsomes (SRM) fuse when incubated with physiological concentrations of GTP and MgCl2. In order to examine further to what extent such fusions are associated with other membrane functions of rough endoplasmic reticulum, we have evaluated the role of cytosolically exposed peptide constituents of SRM in fusion, and the possible relationship of GTP/MgCl2-induced fusion in protein transport across endoplasmic reticulum (ER) membranes, and in ER-Golgi interactions. Controlled proteolytic digestion of SRM led to the loss of fusion capability at 15 micrograms/ml trypsin--a concentration which maintained the latency of intraluminal mannose-6-phosphatase. Hence, a cytosolically exposed protein(s) regulated fusion. Based on ribonuclease-induced ribosome capping experiments, it was further concluded that the cytosolic oriented protein(s) was sequestered beneath the ribosome. As co-translational cell free translocation of placental lactogen across SRM was similar in control membranes compared to those rendered incapable of fusing, it was concluded that the fusion phenomenon may not be related to translocation. Under conditions promoting homologous fusion of SRM or Golgi membranes, mixtures of the two membranes showed no heterologous membrane fusion as assessed morphologically or by the transport of newly synthesized membrane glycoprotein. These experiments attest to the specificity of cytosolically exposed protein(s) in regulating nucleotide/divalent cation-induced membrane fusion.     

Engineering of vesicle trafficking improves heterologous protein secretion in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often restricted due to the limitations of the host strain. In the protein secretory pathway, the protein trafficking between different organelles is catalyzed by the soluble NSF (N-ethylmaleimide-sensitive factor) receptor (SNARE) complex and regulated by the Sec1/Munc18 (SM) proteins. In this study, we report that over-expression of the SM protein encoding genes SEC1 and SLY1, improves the protein secretion in S. cerevisiae. Engineering Sec1p, the SM protein that is involved in vesicle trafficking from Golgi to cell membrane, improves the secretion of heterologous proteins human insulin precursor and α-amylase, and also the secretion of an endogenous protein invertase. Enhancing Sly1p, the SM protein regulating the vesicle fusion from endoplasmic reticulum (ER) to Golgi, increases α-amylase production only. Our study demonstrates that strengthening the protein trafficking in ER-to-Golgi and Golgi-to-plasma membrane process is a novel secretory engineering strategy for improving heterologous protein production in S. cerevisiae.     

Rab1 interaction with a GM130 effector complex regulates COPII vesicle cis--Golgi tethering

Members of the Rab family of small molecular weight GTPases regulate the fusion of transport intermediates to target membranes along the biosynthetic and endocytic pathways. We recently demonstrated that Rab1 recruitment of the tethering factor p115 into a cis -SNARE complex programs coat protein II vesicles budding from the endoplasmic reticulum (donor compartment) for fusion with the Golgi apparatus (acceptor compartment) (Allan BB, Moyer BD, Balch WE. Science 2000; 289: 444–448). However, the molecular mechanism(s) of Rab regulation of Golgi acceptor compartment function in endoplasmic reticulum to Golgi transport are unknown. Here, we demonstrate that the cis -Golgi tethering protein GM130, complexed with GRASP65 and other proteins, forms a novel Rab1 effector complex that interacts with activated Rab1-GTP in a p115-independent manner and is required for coat protein II vesicle targeting/fusion with the cis -Golgi. We propose a 'homing hypothesis' in which the same Rab interacts with distinct tethering factors at donor and acceptor membranes to program heterotypic membrane fusion events between transport intermediates and their target compartments.     

Membrane topology and function of Der3/Hrd1p as a ubiquitin-protein ligase (E3) involved in endoplasmic reticulum degradation

The endoplasmic reticulum contains a protein quality control system that discovers malfolded or unassembled secretory proteins and subjects them to degradation in the cytosol. This requires retrograde transport of the respective proteins from the endoplasmic reticulum back to the cytosol via the Sec61 translocon. In addition, a fully competent ubiquitination machinery and the 26 S proteasome are necessary for retrotranslocation and degradation. Ubiquitination of mutated and malfolded proteins of the endoplasmic reticulum is dependent mainly on the ubiquitin-conjugating enzyme Ubc7p. In addition, several new membrane components of the endoplasmic reticulum are required for degradation. Here we present the topology of the previously discovered RING-H2 finger protein Der3/Hrd1p, one of the new components of the endoplasmic reticulum membrane. The protein spans the membrane six times. The amino terminus and the carboxyl terminus containing the RING finger domain face the cytoplasm. Altogether, RING finger-dependent ubiquitination of malfolded carboxypeptidase yscY in vivo, as well as of Der3/Hrd1p itself in vitro and RING finger-dependent binding of Ubc7p, uncovers Der3/Hrd1p as the ubiquitin-protein ligase (E3) of the endoplasmic reticulum-associated protein degradation process.     

Persistence of Golgi matrix distribution exhibits the same dependence on Sar1p activity as a Golgi glycosyltransferase

We investigated the relative distributional persistence of Golgi 'matrix' proteins and glycosyltransferases to an endoplasmic reticulum exit block induced by expression of a GDP-restricted Sar1p. HeLa cells were microinjected with plasmid encoding the GDP-restricted mutant (T39N) of Sar1p to block endoplasmic reticulum exit and then scored for the distribution of GM130 (Golgi m atrix protein of 130  kDa), a cis located golgin; p27, a member of the p24 family of proteins; giantin, a protein that interacts indirectly with GM130; and the Golgi glycosyltransferase, N-acetylgalactosaminyltransferase-2 (GalNAcT2). All of these proteins lost their compact, juxtanuclear distribution and displayed characteristics of endoplasmic reticulum/cytoplasmic accumulation with the same dependence on plasmid concentration. The kinetics of redistribution of GM130 and GalNAcT2 were identical. Expression of Sar1pT39N displaced the COPII coat protein Sec13p from endoplasmic reticulum exit sites consistent with disruption of these sites. This occurred without disturbing the overall distribution of endoplasmic reticulum membrane. Furthermore, the reassembly of a juxtanuclear Golgi matrix as assayed by the distribution of GM130 following washout of the Golgi disrupting drug, brefeldin A, was blocked by microinjected Sar1pT39N plasmids. We conclude that the persistence, i.e. stability and maintenance, of Golgi matrix distribution and its reassembly following drug disruption are exquisitely dependent on Sar1p activity.     

Structural and Functional Analysis of the Globular Head Domain of p115 Provides Insight into Membrane Tethering

Molecular tethers have a central role in the organization of the complex membrane architecture of eukaryotic cells. p115 is a ubiquitous, essential tether involved in vesicle transport and the structural organization of the exocytic pathway. We describe two crystal structures of the N-terminal domain of p115 at 2.0 Å resolution. The p115 structures show a novel α-solenoid architecture constructed of 12 armadillo-like, tether-repeat, α-helical tripod motifs. We find that the H1 TR binds the Rab1 GTPase involved in endoplasmic reticulum to Golgi transport. Mutation of the H1 motif results in the dominant negative inhibition of endoplasmic reticulum to Golgi trafficking. We propose that the H1 helical tripod contributes to the assembly of Rab-dependent complexes responsible for the tether and SNARE-dependent fusion of membranes.     

Folding of viral envelope glycoproteins in the endoplasmic reticulum

Viral glycoproteins fold and oligomerize in the endoplasmic reticulum of the host cell. They employ the cellular machinery and receive assistance from cellular folding factors. During the folding process, they are retained in the compartment and their structural quality is checked by the quality control system of the endoplasmic reticulum. A special characteristic that distinguishes viral fusion proteins from most cellular proteins is the extensive conformational change they undergo during fusion of the viral and cellular membrane. Many viral proteins fold in conjunction with and dependent on a viral partner protein, sometimes even synthesized from the same mRNA. Relevant for folding is that viral glycoproteins from the same or related virus families may consist of overlapping sets of domain modules. The consequences of these features for viral protein folding are at the heart of this review.