Degradation of HMG-CoA reductase in vitro. Cleavage in the membrane domain by a membrane-bound cysteine protease

We have recently shown that the endoplasmic reticulum (ER) membrane protein, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is cleaved in isolated membrane fractions enriched for endoplasmic reticulum. Importantly, the cleavage rate is accelerated when the membranes are prepared from cells that have been pretreated with mevalonate or sterols, physiological regulators of the degradation process in vivo (McGee, T. P., Cheng, H. H., Kumagai, H., Omura, S., and Simoni, R. D. (1996) J. Biol. Chem. 271, 25630-25638). In the current study, we further characterize this in vitro cleavage of HMG-CoA reductase. E64, a specific inhibitor of cysteine-proteases, inhibits HMG-CoA reductase cleavage in vitro. In contrast, lactacystin, an inhibitor of the proteasome, inhibits HMG-CoA reductase degradation in vivo but does not inhibit the in vitro cleavage. Purified ER fractions contain lactacystin-sensitive and E64-insensitive proteasome activity as measured by succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin hydrolysis. We removed the proteasome from purified ER fractions by solubilization with heptylthioglucoside and observed that the detergent extracted, proteasome-depleted membrane fractions retain regulated cleavage of HMG-CoA reductase. This indicates that ER-associated proteasome is not involved in degradation of HMG-CoA reductase in vitro. In order to determine the site(s) of proteolysis of HMG-CoA reductase in vitro, four antisera were prepared against peptide sequences representing various domains of HMG-CoA reductase and used for detection of proteolytic intermediates. The sizes and antibody reactivity of the intermediates suggest that HMG-CoA reductase is cleaved in the in vitro degradation system near the span 8 membrane region, which links the N-terminal membrane domain to the C-terminal catalytic domain of the protein. We conclude that HMG-CoA reductase can be cleaved in the membrane-span 8 region by a cysteine protease(s) tightly associated with ER membranes.     

Cytosolic degradation of T-cell receptor alpha chains by the proteasome

The T-cell antigen receptor (TCR) is an hetero-oligomeric membrane complex composed of at least seven transmembrane polypeptide chains that has served as a model for the assembly and degradation of integral membrane proteins in the endoplasmic reticulum (ER). Unassembled TCRalpha chains fail to mature to the Golgi apparatus and are rapidly degraded by a non-lysosomal "ER degradation" pathway that has been proposed to be autonomous to the ER. In these studies we show that the degradation of core-glycosylated TCRalpha is blocked by N-acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN) and lactacystin, implicating the proteasome in ER degradation. Either acute or chronic treatment of TCRalpha-transfected cells with proteasome inhibitors cause the core-glycosylated TCRalpha chains to progressively shift to an approximately 28-kDa form that lacks N-linked oligosaccharides and the N-terminal signal peptide. The susceptibility of this 28-kDa species to extravesicular protease indicates that it is not protected by the ER membrane and, hence, cytoplasmic. These data suggest a model in which TCRalpha chains that are translocated across the membrane, core-glycosylated, but fail to assemble are dislocated back to the cytoplasm for degradation by cytoplasmic proteasomes. Our data also suggest that covalent modification of TCRalpha with ubiquitin is not required for its degradation.     

Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome

Maturation of wild-type CFTR nascent chains at the endoplasmic reticulum (ER) occurs inefficiently; many disease-associated mutant forms do not mature but instead are eliminated by proteolysis involving the cytosolic proteasome. Although calnexin binds nascent CFTR via its oligosaccharide chains in the ER lumen and Hsp70 binds CFTR cytoplasmic domains, perturbation of these interactions alone is without major influence on maturation or degradation. We show that the ansamysin drugs, geldanamycin and herbimycin A, which inhibit the assembly of some signaling molecules by binding to specific sites on Hsp90 in the cytosol or Grp94 in the ER lumen, block the maturation of nascent CFTR and accelerate its degradation. The immature CFTR molecule was detected in association with Hsp90 but not with Grp94, and geldanamycin prevented the Hsp90 association. The drug-enhanced degradation was decreased by lactacystin and other proteasome inhibitors. Therefore, consistent with other examples of countervailing effects of Hsp90 and the proteasome, it would seem that this chaperone may normally contribute to CFTR folding and, when this function is interfered with by an ansamycin, there is a further shift to proteolytic degradation. This is the first direct evidence of a role for Hsp90 in the maturation of a newly synthesized integral membrane protein by interaction with its cytoplasmic domains on the ER surface.     

Ste6p mutants defective in exit from the endoplasmic reticulum (ER) reveal aspects of an ER quality control pathway in Saccharomyces cerevisiae

We are studying the intracellular trafficking of the multispanning membrane protein Ste6p, the a-factor transporter in Saccharomyces cerevisiae and a member of the ATP-binding cassette superfamily of proteins. In the present study, we have used Ste6p as model for studying the process of endoplasmic reticulum (ER) quality control, about which relatively little is known in yeast. We have identified three mutant forms of Ste6p that are aberrantly ER retained, as determined by immunofluorescence and subcellular fractionation. By pulse-chase metabolic labeling, we demonstrate that these mutants define two distinct classes. The single member of Class I, Ste6-166p, is highly unstable. We show that its degradation involves the ubiquitin-proteasome system, as indicated by its in vivo stabilization in certain ubiquitin-proteasome mutants or when cells are treated with the proteasome inhibitor drug MG132. The two Class II mutant proteins, Ste6-13p and Ste6-90p, are hyperstable relative to wild-type Ste6p and accumulate in the ER membrane. This represents the first report of a single protein in yeast for which distinct mutant forms can be channeled to different outcomes by the ER quality control system. We propose that these two classes of ER-retained Ste6p mutants may define distinct checkpoint steps in a linear pathway of ER quality control in yeast. In addition, a screen for high-copy suppressors of the mating defect of one of the ER-retained ste6 mutants has identified a proteasome subunit, Hrd2p/p97, previously implicated in the regulated degradation of wild-type hydroxymethylglutaryl-CoA reductase in the ER membrane.     

CD4 glycoprotein degradation induced by human immunodeficiency virus type 1 Vpu protein requires the function of proteasomes and the ubiquitin-conjugating pathway

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a type I anchored integral membrane phosphoprotein with two independent functions. First, it regulates virus release from a post-endoplasmic reticulum (ER) compartment by an ion channel activity mediated by its transmembrane anchor. Second, it induces the selective down regulation of host cell receptor proteins (CD4 and major histocompatibility complex class I molecules) in a process involving its phosphorylated cytoplasmic tail. In the present work, we show that the Vpu-induced proteolysis of nascent CD4 can be completely blocked by peptide aldehydes that act as competitive inhibitors of proteasome function and also by lactacystin, which blocks proteasome activity by covalently binding to the catalytic beta subunits of proteasomes. The sensitivity of Vpu-induced CD4 degradation to proteasome inhibitors paralleled the inhibition of proteasome degradation of a model ubiquitinated substrate. Characterization of CD4-associated oligosaccharides indicated that CD4 rescued from Vpu-induced degradation by proteasome inhibitors is exported from the ER to the Golgi complex. This finding suggests that retranslocation of CD4 from the ER to the cytosol may be coupled to its proteasomal degradation. CD4 degradation mediated by Vpu does not require the ER chaperone calnexin and is dependent on an intact ubiquitin-conjugating system. This was demonstrated by inhibition of CD4 degradation (i) in cells expressing a thermally inactivated form of the ubiquitin-activating enzyme E1 or (ii) following expression of a mutant form of ubiquitin (Lys48 mutated to Arg48) known to compromise ubiquitin targeting by interfering with the formation of polyubiquitin complexes. CD4 degradation was also prevented by altering the four Lys residues in its cytosolic domain to Arg, suggesting a role for ubiquitination of one or more of these residues in the process of degradation. The results clearly demonstrate a role for the cytosolic ubiquitin-proteasome pathway in the process of Vpu-induced CD4 degradation. In contrast to other viral proteins (human cytomegalovirus US2 and US11), however, whose translocation of host ER molecules into the cytosol occurs in the presence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked.     

Regulated Co-translational ubiquitination of apolipoprotein B100. A new paradigm for proteasomal degradation of a secretory protein

Presentation of a wild-type secretory protein, apolipoprotein B100 (apoB), to the cytosol for ubiquitin-proteasome proteolysis has been observed in HepG2 cells. A currently accepted model for proteasomal degradation of secretory proteins is retrograde translocation of the substrate polypeptides from the lumen of endoplasmic reticulum (ER) back to the cytosol. In this report, we present evidence that newly synthesized apoB becomes exposed to the cytosol and targeted to the proteasomes in a co-translational manner. Thus, after protein translation was synchronized with puromycin, partially synthesized apoB polypeptides were found to be conjugated to ubiquitin. The magnitude of co-translational ubiquitination and subsequent degradation of apoB was increased when cells were pretreated with either herbimycin A to induce cytosolic Hsp70 or with an inhibitor of microsomal triglyceride transfer protein; both treatments impede translocation of nascent apoB across the ER membrane. These treatments also decreased secretion of apoB and increased its degradation via the ubiquitin-proteasome pathway. We suggest that translocation arrest with subsequent co-translational exposure to the cytosol provides an alternative model to explain how mammalian secretory proteins can overcome topological segregation by the ER membrane and undergo degradation by the ubiquitin-proteasome pathway.     

Dissociation of proteasomal degradation of biosynthesized viral proteins from generation of MHC class I-associated antigenic peptides

To study the role of proteasomes in Ag presentation, we analyzed the effects of proteasome inhibitors Cbz-Leu-Leu-Leucinal and lactacystin on the ability of mouse fibroblast cells to present recombinant vaccinia virus gene products to MHC class I-restricted T cells. The effects of the inhibitors depended on the determinant analyzed. For influenza virus nucleoprotein (NP), presentation of the immunodominant Kk-restricted determinant (NP(50-57)) was marginally inhibited, whereas presentation of the immunodominant Kd-restricted determinant (NP(147-155)) was enhanced, particularly by lactacystin. Biochemical purification of peptides confirmed that lactacystin enhanced the generation of Kd-NP(147-155) complexes fourfold. Lactacystin also enhanced the recovery of one Kd-restricted vaccinia virus determinant from HPLC fractions, while inhibiting recovery of another. The inhibitors were used at sufficient concentrations to block presentation of biosynthesized full-length OVA and to completely stabilize a rapidly degraded chimeric ubiquitin-NP fusion protein. Strikingly, presentation of antigenic peptides from this protein was unaffected by proteasome inhibitors. We also observed that proteasome inhibitors induced expression of cytosolic and endoplasmic reticulum stress-responsive proteins. These data demonstrate first that the processes of protein degradation and generation of antigenic peptides from cytosolic proteins can be dissociated, and second that effects of proteasome inhibitors on Ag presentation may reflect secondary effects on cellular metabolism.     

Presecretory degradation of apolipoprotein [a] is mediated by the proteasome pathway

Plasma levels of atherogenic lipoprotein [a] (Lp[a]) vary over a 1000-fold range and are largely determined by the gene for its unique glycoprotein, apolipoprotein [a] (apo[a]). The apo[a] locus comprises more than 100 alleles, encoding proteins from <300 to >800 kDa. Using primary baboon hepatocyte cultures, we previously demonstrated that differences in the secretion efficiency of apo[a] allelic variants contribute to the variation in plasma Lp[a] levels. In the current study, we investigated the mechanism of apo[a] presecretory degradation. The proteasome inhibitors, acetyl-leucyl-leucyl-norleucinal and lactacystin, prevented apo[a] degradation and increased apo[a] secretion. Transfection with an HA-tagged ubiquitin construct demonstrated the accumulation of ubiquitinated apo[a] in the presence of lactacystin. These results suggest a role for the cytoplasmic proteasome in apo[a] proteolysis. Apo[a] that accumulated intracellularly in the presence of lactacystin remained sensitive to endo-B-N-glucosaminidase H, and apo[a] degradation was reversibly inhibited by brefeldin A, suggesting that transport to a post-endoplasmic reticulum (ER) pre-medial Golgi compartment is required for apo[a] degradation. Newly synthesized apo[a] bound to the ER chaperone calnexin and conditions that enhanced this interaction prevented apo[a] degradation, suggesting that calnexin can protect apo[a] from proteolysis. These studies provide further support for the role of the proteasome in endoplasmic reticulum quality control, and expand this role to one that influences plasma levels of the atherogenic lipoprotein Lp[a].-White, A. L., B. Guerra, J. Wang, and R. E. Lanford. Presecretory degradation of apolipoprotein[a] is mediated by the proteasome pathway.     

Evidence that endoplasmic reticulum (ER)-associated degradation of cystic fibrosis transmembrane conductance regulator is linked to retrograde translocation from the ER membrane

The ubiquitin-proteasome pathway has been implicated in the degradation of newly synthesized, misfolded and unassembled proteins in the endoplasmic reticulum (ER). Using a cell-free reticulocyte lysate system we have examined the relationship between biosynthesis and ER-associated degradation of the cystic fibrosis transmembrane conductance regulator (CFTR), a polytopic protein with 12 predicted transmembrane segments. Our results provide direct evidence that full-length, glycosylated and membrane-integrated CFTR is a substrate for degradation and that degradation involves polyubiquitination and cytosolic proteolytic activity. CFTR ubiquitination was both temperature- and ATP-dependent. Degradation was significantly inhibited by EDTA, apyrase, and the proteasome inhibitors hemin and MG132. Degradation was inhibited to a lesser extent by clasto-lactacystin beta-lactone, ALLN, and Nalpha-tosyl-L-phenylalanine chloromethyl ketone and was relatively unaffected by lactacystin and N-tosyl lysyl chloromethyl ketone. In the presence of hemin, polyubiquitinated CFTR remained tightly associated with ER microsomes. However, membrane-bound ubiquitinated CFTR could be subsequently degraded into trichloroacetic acid-soluble fragments upon incubation in hemin-free, ATP-containing lysate. Thus ER-associated degradation of CFTR occurs via a membrane-bound, rather than cytosolic, intermediate and likely involves recruitment of degradation machinery to the ER membrane. Our data suggest a model in which the degradation of polytopic proteins such as CFTR is coupled to retrograde translocation and removal of the polypeptide from the lipid bilayer.     

Ubiquitination of protein kinase C-alpha and degradation by the proteasome

Bryostatins and phorbol esters acutely activate and subsequently down-regulate protein kinase C (PKC) by inducing its proteolysis via an unknown pathway. Here we show that treatment of renal epithelial cells with bryostatin 1 (Bryo) produced novel PKC-alpha species, which were larger than the native protein (80 kDa). The >80 kDa PKC-alpha species contained Ubi as indicated by immunostaining and accumulated in the presence of lactacystin, a selective inhibitor of proteolysis by the proteasome. In vitro experiments with 125I-ubiquitin and membranes from Bryo-treated cells showed that PKC-alpha became ubiquitinated by a reaction that depended on ATP and a cytosolic fraction. Lactacystin or a peptidyl aldehyde, Bz-Gly-Leu-Ala-leucinal, which inhibits certain proteinase activities of the proteasome, inhibited Bryo-evoked disappearance of PKC-alpha protein from the cells. Lacta preserved Bryo-induced 32P-labeled PKC-alpha indicating that the proteasome inhibitor spared activated enzyme from down-regulation in vivo. These findings show that Bryo induces the degradation of PKC-alpha by the ubiquitin-proteasome complex.     

The degradation of apolipoprotein B100 is mediated by the ubiquitin-proteasome pathway and involves heat shock protein 70

Apolipoprotein B (apoB) is the major protein component of atherogenic lipoproteins of hepatic origin. In HepG2 cells, the standard cell culture model of human hepatic lipoprotein metabolism, there is a limited availability of core lipids in the endoplasmic reticulum for association with nascent apoB. Under these conditions, apoB is partially translocated, interacts with cytosolic Hsp70, and undergoes rapid degradation. We show that increasing the expression of Hsp70 in HepG2 cells promotes apoB degradation. In addition, apoB is polyubiquitinated and its degradation both normally and after Hsp70 induction is blocked by inhibitors of the proteasome. The apoB that accumulates after proteasome inhibition is endoplasmic reticulum-associated and can be assembled into lipoproteins and secreted if new lipid synthesis is stimulated. Thus, apoB is the first example of a wild-type mammalian protein whose secretion is regulated by degradation in the cytosol via the ubiquitin-proteasome pathway. Furthermore, targeting of this secretory protein to the proteasome is regulated by the molecular chaperone Hsp70 and the availability of apoB's lipid-ligands.     

Calpain involvement in calphostin C-induced apoptosis

A major problem in assessing the role of calpains in apoptosis induction concerns the fact that calpain inhibitors can also impair the activity of the proteasome, also reported to be involved in apoptosis. Herein we showed that apoptosis induced by calphostin C in U937 human promonocytic leukemia cells was associated, at its onset, with enhanced protein (poly)ubiquitination. This observation prompted us to study whether protein degradation through the ubiquitin/proteasome pathway was involved in apoptosis induction. We found that N-acetyl-Leu-Leu-norleucinal (50 microM), a proteasome as well as a calpain inhibitor, was able to reduce calphostin C-induced apoptosis by approximately 60%, whereas lactacystin (10 microM), a specific proteasome inhibitor, was ineffective. These results suggest that calphostin C-induced apoptosis is partly calpain-mediated, but does not require protein degradation through the ubiquitin/proteasome pathway.     

The proteasome inhibitor lactacystin prevents the generation of an endoplasmic reticulum leader-derived T cell epitope

The presentation of viral antigens on MHC class I molecules requires their intracellular fragmentation into peptides of appropriate length and anchor residue positions. Evidence has accumulated that the proteasome is the endoprotease in charge of the generation of MHC class I ligands in the cytoplasm. The generation of T cell epitopes derived from the leader peptides of endoplasmic reticulum (ER) targeted proteins, however. has been reported to be independent of the proteasome. Here we show that the H-2Db restricted antigen presentation of the immunodominant T cell epitope derived from the ER leader of the glycoprotein of lymphocytic choriomeningitis virus (LCMV) is completely abolished by administration of the proteasome inhibitor lactacystin. Thus our data support the role of the proteasome in class I restricted antigen processing and extend it to an ER leader derived epitope from a viral glycoprotein.     

The requirement for molecular chaperones during endoplasmic reticulum-associated protein degradation demonstrates that protein export and import are mechanistically distinct

Polypeptide import into the yeast endoplasmic reticulum (ER) requires two hsp70s, Ssa1p in the cytosol and BiP (Kar2p) in the ER lumen. After import, aberrant polypeptides may be exported to the cytoplasm for degradation by the proteasome, and defects in the ER chaperone calnexin (Cne1p) compromise their degradation. Both import and export require BiP and the Sec61p translocation complex, suggesting that import and export may be mechanistically related. We now show that the cne1Delta and two kar2 mutant alleles exhibit a synthetic interaction and that the export and degradation of pro-alpha factor is defective in kar2 mutant microsomes. Pulse-chase analysis indicates that A1PiZ, another substrate for degradation, is stabilized in the kar2 strains at the restrictive temperature. Because two of the kar2 mutants examined are proficient for polypeptide import, the roles of BiP during ER protein export and import differ, indicating that these processes must be mechanistically distinct. To examine whether Ssa1p drives polypeptides from the ER and is also required for degradation, we assembled reactions using strains either containing a mutation in SSA1 or in which the level of Ssa1p could be regulated. We found that pro-alpha factor and A1PiZ were degraded normally, indicating further that import and export are distinct and that other cytosolic factors may pull polypeptides from the ER.     

Characterization of KIF1C, a new kinesin-like protein involved in vesicle transport from the Golgi apparatus to the endoplasmic reticulum

Kinesins comprise a large family of microtubule-based motor proteins, of which individual members mediate specific types of motile processes. Using the ezrin domain of the protein-tyrosine phosphatase PTPD1 as a bait in a yeast two-hybrid screen, we identified a new kinesin-like protein, KIF1C. KIF1C represents a member of the Unc104 subfamily of kinesin-like proteins that are involved in the transport of mitochondria or synaptic vesicles in axons. Like its homologues, the 1103-amino acid protein KIF1C consists of an amino-terminal motor domain followed by a U104 domain and probably binds to target membranes through carboxyl-terminal sequences. Interestingly, KIF1C was tyrosine-phosphorylated after peroxovanadate stimulation when overexpressed in 293 or NIH3T3 fibroblasts or in native C2C12 cells. Using immunofluorescence, we found that KIF1C is localized primarily at the Golgi apparatus. In brefeldin A-treated cells, the Golgi membranes and KIF1C redistributed to the endoplasmic reticulum (ER). This brefeldin A-induced flow of Golgi membranes into the ER was inhibited in cells transiently overexpressing catalytically inactive KIF1C. In conclusion, our data suggest an involvement of tyrosine phosphorylation in the regulation of the Golgi to ER membrane flow and describe a new kinesin-like motor protein responsible for this transport.     

The effect of the proteasome inhibitor lactacystin on the presentation of transporter associated with antigen processing (TAP)-dependent and TAP-independent peptide epitopes by class I molecules

Cells were treated with two proteolytic inhibitors, N-acetyl-leucyl-leucyl-norleucinal and lactacystin, the latter reported to be a specific inhibitor for the proteasome. Both inhibitors retarded the maturation of endo-H-resistant forms of murine and human class I molecules from their endo-H-sensitive precursors in cell lines with functional TAP proteins. HLA-A2 maturation readily occurs in TAP-deficient T2 cells, and it has been shown that the peptides associated with A2 are derived from the leader segment of proteins in the secretory pathway. This maturation is inhibited by N-acetyl-leucyl-leucyl-norleucinal but not lactacystin, indicating that the proteasome is not required for the generation of HLA-A2 binding peptides in these cells. The murine class Ib molecule Qa-1b presents a leader peptide derived from D-end class I molecules to alloreactive CTL. Since this presentation is dependent on the expression of TAP proteins, we determined if this requirement reflects a need for the proteasome to process this peptide. We found that lactacystin did not inhibit the maturation of endo-H-resistant forms of Qa-1b that are dependent on this leader peptide for its maturation, nor did it inhibit the expression of this peptide-Qa-1b complex in a functional assay. Thus, unlike conventional cytosolic peptides, leader peptides (regardless of whether they are dependent on TAP for their presentation) do not require the proteasome for processing.     

Ubiquitination is required for the retro-translocation of a short-lived luminal endoplasmic reticulum glycoprotein to the cytosol for degradation by the proteasome

In the endoplasmic reticulum (ER), an efficient "quality control system" operates to ensure that mutated and incorrectly folded proteins are selectively degraded. We are studying ER-associated degradation using a truncated variant of the rough ER-specific type I transmembrane glycoprotein, ribophorin I. The truncated polypeptide (RI332) consists of only the 332 amino-terminal amino acids of the protein corresponding to most of its luminal domain and, in contrast to the long-lived endogenous ribophorin I, is rapidly degraded. Here we show that the ubiquitin-proteasome pathway is involved in the destruction of the truncated ribophorin I. Thus, when RI332 that itself appears to be a substrate for ubiquitination was expressed in a mutant hamster cell line harboring a temperature-sensitive mutation in the ubiquitin-activating enzyme E1 affecting ubiquitin-dependent proteolysis, the protein is dramatically stabilized at the restrictive temperature. Moreover, inhibitors of proteasome function effectively block the degradation of RI332. Cell fractionation experiments indicate that RI332 accumulates in the cytosol when degradation is prevented by proteasome inhibitors but remains associated with the lumen of the ER under ubiquitination-deficient conditions, suggesting that the release of the protein into the cytosol is ubiquitination-dependent. Accordingly, when ubiquitination is impaired, a considerable amount of RI332 binds to the ER chaperone calnexin and to the Sec61 complex that could effect retro-translocation of the polypeptide to the cytosol. Before proteolysis of RI332, its N-linked oligosaccharide is cleaved in two distinct steps, the first of which might occur when the protein is still associated with the ER, as the trimmed glycoprotein intermediate efficiently interacts with calnexin and Sec61. From our data we conclude that the steps that lead a newly synthesized luminal ER glycoprotein to degradation by the proteasome are tightly coupled and that especially ubiquitination plays a crucial role in the retro-translocation of the substrate protein for proteolysis to the cytosol.     

PIG-B, a membrane protein of the endoplasmic reticulum with a large lumenal domain, is involved in transferring the third mannose of the GPI anchor

Many eukaryotic cell surface proteins are bound to the membrane via the glycosylphosphatidylinositol (GPI) anchor that is covalently linked to their carboxy-terminus. The GPI anchor precursor is synthesized in the endoplasmic reticulum (ER) and post-translationally linked to protein. We cloned a human gene termed PIG-B (phosphatidylinositol glycan of complementation class B) that is involved in transferring the third mannose. PIG-B encodes a 554 amino acid, ER transmembrane protein with an amino-terminal portion of approximately 60 amino acids on the cytoplasmic side and a large carboxy-terminal portion of 470 amino acids within the ER lumen. A mutant PIG-B lacking the cytoplasmic portion remains active, indicating that the functional site of PIG-B resides on the lumenal side of the ER membrane. The PIG-B gene was localized to chromosome 15 at q21-q22. This autosomal location would explain why PIG-B is not involved in the defective GPI anchor synthesis in paroxysmal nocturnal hemoglobinuria, which is always caused by a somatic mutation of the X-linked PIG-A gene.     

Dual roles of proteasome in the metabolism of presenilin 1

Presenilin 1 (PS1) has been identified as a causative gene for most early-onset familial Alzheimer's disease. Biochemical studies revealed that PS1 exists predominantly as two processed fragments in cells and brain tissues. We prepared stably transfected cells expressing the wild-type and familial Alzheimer's disease-associated mutants of PS1 and investigated the enzyme that participates in the metabolism of PS1. After treatment of the cells with proteasome inhibitors, the full-length PS1 was significantly accumulated. The levels of N- and C-terminal fragments were also increased. The accumulation of PS1 with a deletion of exon 10, which is unable to be processed, on treatment of the transfected cells with lactacystin indicated that proteasome can degrade full-length PS1. A synthetic peptide that includes the processing region of PS1 was cleaved by 20S proteasome at the putative processing sites after Met288 and Glu299. Metabolic labeling experiments showed that the appearance of the N-terminal fragment was attenuated by the inhibitor. Finally, 28-kDa N- and 20-kDa C-terminal fragments were generated by purified PS1 in vitro. These data indicated that the proteasome pathway is involved in PS1 processing. These results demonstrate that the proteasome pathway plays dual roles in processing and degradation of PS1.     

Changing patterns of localization of the tobacco mosaic virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection

Tobacco mosaic virus (TMV) derivatives that encode movement protein (MP) as a fusion to the green fluorescent protein (MP:GFP) were used in combination with antibody staining to identify host cell components to which MP and replicase accumulate in cells of infected Nicotiana benthamiana leaves and in infected BY-2 protoplasts. MP:GFP and replicase colocalized to the endoplasmic reticulum (ER; especially the cortical ER) and were present in large, irregularly shaped, ER-derived structures that may represent "viral factories." The ER-derived structures required an intact cytoskeleton, and microtubules appeared to redistribute MP:GFP from these sites during late stages of infection. In leaves, MP:GFP accumulated in plasmodesmata, whereas in protoplasts, the MP:GFP was targeted to distinct, punctate sites near the plasma membrane. Treating protoplasts with cytochalasin D and brefeldin A at the time of inoculation prevented the accumulation of MP:GFP at these sites. It is proposed that the punctate sites anchor the cortical ER to plasma membrane and are related to sites at which plasmodesmata form in walled cells. Hairlike structures containing MP:GFP appeared on the surface of some of the infected protoplasts and are reminiscent of similar structures induced by other plant viruses. We present a model that postulates the role of the ER and cytoskeleton in targeting the MP and viral ribonucleoprotein from sites of virus synthesis to the plasmodesmata through which infection is spread.