Cytochromes P450 2C1/2 and P450 2E1 are retained in the endoplasmic reticulum membrane by different mechanisms

Cytochrome P450 (P450) 2C1/2 contains redundant endoplasmic reticulum (ER) retention signals and is excluded from the recycling pathway. Other P450s, such as P450 2E1, have been detected in the plasma membrane and Golgi apparatus. To examine whether the mechanisms of ER retention might differ for P450 2C1/2 and P450 2E1, chimeras of green flourescent protein and the full-length proteins, N-terminal signal/anchor sequences, or the cytoplasmic catalytic domains from these proteins have been expressed in COS1 cells. Chimeras with either the N-terminal signal/anchor sequence or the cytoplasmic domain of P450 2C1/2 were retained in the ER and the distribution was not altered by treatment with nocodazole. A chimera with full-length P450 2E1 was located in the ER, but in contrast to P450 2C1/2, treatment with nocodazole resulted in redistribution to a vesicular pattern, which suggested that this protein was retained in the ER by a retrieval mechanism. In support of this possibility, the P450 2E1 chimera, but not the P450 2C1/2 chimera, was included in transport vesicles generated in an in vitro budding assay. A chimera with only the N-terminal signal/anchor sequence of P450 2E1 fused to green fluorescent protein was located in the ER and nocodazole treatment altered its distribution, whereas a chimera with only the cytoplasmic domain of P450 2E1 was not efficiently retained in the ER and accumulated primarily in the Golgi region. These results demonstrate that the mechanisms for retention in the ER of two closely related members of the P450 superfamily are different and that the N-terminal signal/anchor sequence contains the dominant retention signal.     

The transmembrane domain of murine alpha-mannosidase IB is a major determinant of Golgi localization

Murine alpha1,2-mannosidase IB is a type II transmembrane protein localized to the Golgi apparatus where it is involved in the biogenesis of complex and hybrid N-glycans. This enzyme consists of a cytoplasmic tail, a transmembrane domain followed by a "stem" region and a large C-terminal catalytic domain. To analyze the determinants of targeting, we constructed various deletion mutants of murine alpha1,2-mannosidase IB as well as alpha1,2-mannosidase IB/yeast alpha1,2-mannosidase and alpha1,2-mannosidase IB/GFP chimeras and localized these proteins by fluorescence microscopy, when expressed transiently in COS7 cells. Replacing the catalytic domain of alpha1,2-mannosidase IB with that of the homologous yeast alpha1,2-mannosidase and deleting the "stem" region in this chimera had no effect on Golgi targeting, but caused increased cell surface localization. The N-terminal tagged protein lacking a catalytic domain was also localized to the Golgi. In the latter case, when the stem region was partially or completely removed, the protein was found in both the ER and the Golgi. A chimera consisting of the alpha1,2-mannosidase IB N-terminal region (cytoplasmic and transmembrane domains plus 10 amino acids of the "stem" region) and GFP was localized mainly to the Golgi. Deletion of 30 out of 35 amino acids in the cytoplasmic tail had no effect on Golgi localization. A GFP chimera lacking the entire cytoplasmic tail was found in both the ER and the Golgi. These results indicate that the transmembrane domain of alpha1,2-mannosidase IB is a major determinant of Golgi localization.     

人朊蛋白不同N-糖基化修饰突变体的构建及其在哺乳动物细胞中表达

构建并表达人朊蛋白N-糖基化修饰位点突变的真核表达载体,有助于进一步研究朊蛋白N-糖基化修饰的生物学功能。定点突变野生型人朊蛋白基因PRNP,将获得的突变体亚克隆至真核表达载体pcDNA3.1中,并在人宫颈癌细胞株HeLa中瞬时表达各种朊蛋白糖基化修饰位点突变体,利用免疫印迹和糖苷酶消化等糖蛋白分析方法鉴定表达产物的糖基化形式。经Western blot鉴定,野生型和突变型朊蛋白表达产物出现不同形式的泳动特征,分别出现特异性糖基化修饰的多个条带,单糖基化修饰的两条条带和无糖基化修饰的一条条带。经PNGase F糖苷酶消化,野生型和糖基化单点突变型表达产物均能被糖苷酶消化,其分子量下移,去糖基化突变型表达产物的分子条带位置不变。通过突变野生型人朊蛋白基因PRNP的N-糖基化修饰位点,获得单糖基化修饰和去N-糖基化修饰的6种人朊蛋白突变体,并能够在HeLa细胞株中瞬时表达单糖基化修饰和去N-糖基化修饰朊蛋白,为进一步研究朊蛋白的相关功能建立良好基础。     

Fluorescence resonance energy transfer analysis of cytochromes P450 2C2 and 2E1 molecular interactions in living cells

The molecular organization of microsomal cytochromes P450 (P450s) and formation of complexes with P450 reductase have been studied previously with isolated proteins and in reconstituted systems. Although these studies demonstrated that some P450s oligomerize in vitro, neither oligomerization nor interactions of P450 with P450 reductase have been studied in living cells. Here we have used fluorescence resonance energy transfer (FRET) to study P450 oligomerization and binding to P450 reductase in live transfected cells. Cytochrome P450 2C2, but not P450 2E1, forms homo-oligomeric structures, and this self-association is mediated by the signal-anchor sequence. Because P450 2C2, in contrast to P450 2E1, is directly retained in the endoplasmic reticulum (ER), these results could suggest that oligomerization may prevent transport from the ER. However, P450 2C1 signal-anchor sequence chimera defective in ER retention also formed oligomers, and chimera containing the cytoplasmic domain of P450 2C2, which is directly retained in the ER, did not exhibit self-oligomerization, which indicates that oligomerization is not correlated with direct retention. By using FRET, we have also detected binding of P450 2C2 and P450 2E1 to P450 reductase. In contrast to self-oligomerization, the catalytic domain can mediate an interaction of P450 2C2 with P450 reductase. These results suggest that microsomal P450s may differ in their quaternary structure but that these differences do not detectably affect interaction with the reductase or transport from the ER.     

Altered glycosylation sites of the delta subunit of the acetylcholine receptor (AChR) reduce alpha delta association and receptor assembly.

We have used mutagenesis to investigate the potential N-glycosylation sites in the delta subunit of the mouse muscle acetylcholine receptor (AChR). Of the three sites, Asn76, Asn143, and Asn169, only the first two were glycosylated when the delta subunit was expressed in COS cells. Because the heterologously expressed delta subunit was similar in its properties to that expressed in C2 muscle cells, the sites of glycosylation are likely to be the same in both cases. In COS cells, mutations of the delta subunit that prevented glycosylation at either of the sites did not change its metabolic stability nor its steady-state level. These results are in contrast to those found previously for the alpha subunit, in which glycosylation at a single site metabolically stabilized the polypeptide (Blount, P., and Merlie, J. P. (1990) J. Cell Biol. 111, 2613-2622). Mutations of the delta subunit that prevented glycosylation, however, decreased its ability to form an alpha delta heterodimer when the alpha and delta subunit were expressed together. When all four subunits of the AChR (alpha, beta, delta, and epsilon) were coexpressed, mutation of the delta subunit to prevent glycosylation resulted in a reduced amount of fully assembled AChR and reduced surface AChR levels, consistent with the role of the heterodimer in the assembly reaction. These results suggest that glycosylation of the delta subunit at both Asn76 and Asn143 is needed for its efficient folding and/or its subsequent interaction with the alpha subunit.     

Epithelial sodium channel exit from the endoplasmic reticulum is regulated by a signal within the carboxyl cytoplasmic domain of the alpha subunit

Epithelial sodium channels (ENaCs) are assembled in the endoplasmic reticulum (ER) from alpha, beta, and gamma subunits, each with two transmembrane domains, a large extracellular loop, and cytoplasmic amino and carboxyl termini. ENaC maturation involves transit through the Golgi complex where Asn-linked glycans are processed to complex type and the channel is activated by furin-dependent cleavage of the alpha and gamma subunits. To identify signals in ENaC for ER retention/retrieval or ER exit/release, chimera were prepared with the interleukin alpha subunit (Tac) and each of the three cytoplasmic carboxyl termini of mouse ENaC (Tac-Ct) or with gamma-glutamyltranspeptidase and each of the three cytoplasmic amino termini (Nt-GGT). By monitoring acquisition of endoglycosidase H resistance after metabolic labeling, we found no evidence of ER retention of any chimera when compared with control Tac or GGT, but we did observe enhanced exit of Tac-alphaCt when compared with Tac. ER exit of ENaC was assayed after metabolic labeling by following the appearance of cleaved alpha as cleaved alpha subunit, but not non-cleaved alpha, is endoglycosidase H-resistant. Interestingly ER exit of epitope-tagged and truncated alpha (alphaDelta624-699-V5) with full-length betagamma was similar to wild type alpha (+betagamma), whereas ER exit of ENaC lacking the entire cytoplasmic carboxyl tail of alpha (alphaDelta613-699-V5 +betagamma) was significantly reduced. Subsequent analysis of ER exit for ENaCs with mutations within the intervening sequence (613)HRFRSRYWSPG(623) within the context of the full-length alpha revealed that mutation alphaRSRYW(620) to AAAAA significantly reduced ER exit. These data indicate that ER exit of ENaC is regulated by a signal within the alpha subunit carboxyl cytoplasmic tail.     

Cysteineless non-glycosylated monomeric blue fluorescent protein,secBFP2, for studies in the eukaryotic secretory pathway

Fluorescent protein (FP) technologies suitable for use within the eukaryotic secretory pathway are essential for live cell and protein dynamic studies. Localization of FPs within the endoplasmic reticulum (ER) lumen has potentially significant consequences for FP function. All FPs are resident cytoplasmic proteins and have rarely been evolved for the chemically distinct environment of the ER lumen. In contrast to the cytoplasm, the ER lumen is oxidizing and the site where secretory proteins are post-translationally modified by disulfide bond formation and N-glycosylation on select asparagine residues. Cysteine residues and N-linked glycosylation consensus sequences were identified within many commonly utilized FPs. Here, we report mTagBFP is post-translationally modified when localized to the ER lumen. Our findings suggest these modifications can grossly affect the sensitivity and reliability of FP tools within the secretory pathway. To optimize tools for studying events in this important intracellular environment, we modified mTagBFP by mutating its cysteines and consensus N-glycosylation sites. We report successful creation of a secretory pathway-optimized blue FP, secBFP2.     

Signal-mediated retrieval of a membrane protein from the Golgi to the ER in yeast

The Saccharomyces cerevisiae Wbp1 protein is an endoplasmic reticulum (ER), type I transmembrane protein which contains a cytoplasmic dilysine (KKXX) motif. This motif has previously been shown to direct Golgi-to-ER retrieval of type I membrane proteins in mammalian cells (Jackson, M. R., T. Nilsson, and P. A. Peterson. 1993. J. Cell Biol. 121: 317-333). To analyze the role of this motif in yeast, we constructed a SUC2-WBP1 chimera consisting of the coding sequence for the normally secreted glycoprotein invertase fused to the coding sequence of the COOH terminus (including the transmembrane domain and 16-amino acid cytoplasmic tail) of Wbplp. Carbohydrate analysis of the invertase-Wbp1 fusion protein using mannose linkage-specific antiserum demonstrated that the fusion protein was efficiently modified by the early Golgi initial alpha 1,6 mannosyltransferase (Och1p). Subcellular fractionation revealed that > 90% of the alpha 1,6 mannose-modified fusion protein colocalized with the ER (Wbp1p) and not with the Golgi Och1p-containing compartment or other membrane fractions. Amino acid changes within the dily sine motif (KK-->QK, KQ, or QQ) did not change the kinetics of initial alpha 1,6 mannose modification of the fusion protein but did dramatically increase the rate of modification by more distal Golgi (elongating alpha 1,6 and alpha 1,3) mannosyltransferases. These mutant fusion proteins were then delivered directly from a late Golgi compartment to the vacuole, where they were proteolytically cleaved in a PEP4-dependent manner. While amino acids surrounding the dilysine motif played only a minor role in retention ability, mutations that altered the position of the lysines relative to the COOH terminus of the fusion protein also yielded a dramatic defect in ER retention. Collectively, our results indicate that the KKXX motif does not simply retain proteins in the ER but rather directs their rapid retrieval from a novel, Och1p-containing early Golgi compartment. Similar to observations in mammalian cells, it is the presence of two lysine residues at the appropriate COOH-terminal position which represents the most important features of this sorting determinant.     

Plant N-glycan processing enzymes employ different targeting mechanisms for their spatial arrangement along the secretory pathway

The processing of N-linked oligosaccharides in the secretory pathway requires the sequential action of a number of glycosidases and glycosyltransferases. We studied the spatial distribution of several type II membrane-bound enzymes from Glycine max, Arabidopsis thaliana, and Nicotiana tabacum. Glucosidase I (GCSI) localized to the endoplasmic reticulum (ER), alpha-1,2 mannosidase I (ManI) and N-acetylglucosaminyltransferase I (GNTI) both targeted to the ER and Golgi, and beta-1,2 xylosyltransferase localized exclusively to Golgi stacks, corresponding to the order of expected function. ManI deletion constructs revealed that the ManI transmembrane domain (TMD) contains all necessary targeting information. Likewise, GNTI truncations showed that this could apply to other type II enzymes. A green fluorescent protein chimera with ManI TMD, lengthened by duplicating its last seven amino acids, localized exclusively to the Golgi and colocalized with a trans-Golgi marker (ST52-mRFP), suggesting roles for protein-lipid interactions in ManI targeting. However, the TMD lengths of other plant glycosylation enzymes indicate that this mechanism cannot apply to all enzymes in the pathway. In fact, removal of the first 11 amino acids of the GCSI cytoplasmic tail resulted in relocalization from the ER to the Golgi, suggesting a targeting mechanism relying on protein-protein interactions. We conclude that the localization of N-glycan processing enzymes corresponds to an assembly line in the early secretory pathway and depends on both TMD length and signals in the cytoplasmic tail.     

Functional expression of bovine 17 alpha-hydroxylase in COS 1 cells is dependent upon the presence of an amino-terminal signal anchor sequence.

Microsomal forms of eukaryotic cytochrome P450 proteins are integral membrane proteins of the endoplasmic reticulum (ER) membrane which are targeted to the ER via the signal recognition particle pathway. A hydrophobic amino terminus serves as a combined signal sequence and major membrane anchor (signal-anchor sequence) for the microsomal P450s. We have examined the insertion of bovine 17 alpha-hydroxylase (P45017 alpha) into the ER of COS 1 cells in order to evaluate the role of membrane insertion of the amino-terminal signal-anchor of microsomal P450s as a functional determinant for these enzymes. Previously, we have shown that deletion of the hydrophobic amino terminus from P45017 alpha reduced membrane targeting and insertion by 5-fold compared with the wild-type protein, abolished enzymatic activity, and resulted in an aberrant CO difference spectrum. In the present study we have replaced the amino terminus of P45017 alpha with two heterologous signal-anchor sequences, one that is similar and one that is very different from the P45017 alpha sequence. The chimeric proteins were expressed in COS 1 cells. Immunoblot analysis of isolated microsomal membranes show that the heterologous signal-anchor sequences functioned to target the P45017 alpha protein to the ER. Enzymatic assays in intact COS 1 cells indicate that both the chimeric proteins are efficient 17 alpha-hydroxylase enzymes. The amino terminus of P45017 alpha was also replaced with a sequence that is not a signal-anchor, and the expressed protein was neither targeted to the ER nor was functional in COS 1 cells. In conclusion, both the structure and catalytic activity of P45017 alpha in COS 1 cells is dependent upon an amino-terminal sequence that functions as a signal-anchor sequence and not upon the precise sequence of the amino terminus.     

Asparagine-linked glycosylation of human chymotrypsin C is required for folding and secretion but not for enzyme activity

Human chymotrypsin C (CTRC) plays a protective role in the pancreas by mitigating premature trypsinogen activation through degradation. Mutations that abolish activity or secretion of CTRC increase the risk for chronic pancreatitis. The aim of the present study was to determine whether human CTRC undergoes asparagine-linked (N-linked) glycosylation and to examine the role of this modification in CTRC folding and function. We abolished potential sites of N-linked glycosylation (Asn-Xaa-Ser/Thr) in human CTRC by mutating the Asn residues to Ser individually or in combination, expressed the CTRC mutants in HEK 293T cells and determined their glycosylation state using PNGase F and endo H digestion. We found that human CTRC contains a single N-linked glycan on Asn52. Elimination of N-glycosylation by mutation of Asn52 (N52S) reduced CTRC secretion about 10-fold from HEK 293T cells but had no effect on CTRC activity or inhibitor binding. Overexpression of the N52S CTRC mutant elicited endoplasmic reticulum stress in AR42J acinar cells, indicating that N-glycosylation is required for folding of human CTRC. Despite its important role, Asn52 is poorly conserved in other mammalian CTRC orthologs, including the rat which is monoglycosylated on Asn90. Introduction of the Asn90 site in a non-glycosylated human CTRC mutant restored full glycosylation but only partially rescued the secretion defect. We conclude that N-linked glycosylation of human CTRC is required for efficient folding and secretion; however, the N-linked glycan is unimportant for enzyme activity or inhibitor binding. The position of the N-linked glycan is critical for optimal folding, and it may vary among the otherwise highly homologous mammalian CTRC sequences.     

Heteromeric complex formation between CYP2E1 and CYP1A2: evidence for the involvement of electrostatic interactions

Mixed reconstituted systems containing CYP2B4, CYP1A2, and NADPH-cytochrome P450 reductase were previously shown to exhibit a dramatic inhibition of 7-pentoxyresorufin O-dealkylation (PROD) when compared to simple reconstituted systems containing reductase and a single P450 enzyme, results consistent with the formation of CYP1A2-CYP2B4 complexes where the reductase binds with high affinity to the CYP1A2 moiety of the complex. In this report, we provide evidence for an interaction between CYP1A2 and CYP2E1. Synergism of 7-ethoxyresorufin O-deethylation (EROD) and PROD was observed when these P450s were combined in mixed reconstituted systems at subsaturating reductase concentrations. Higher ionic strength attenuated the synergistic stimulation of both PROD and EROD in mixed reconstituted systems, consistent with disruption of heteromeric CYP2E1-CYP1A2 complexes. The effect of ionic strength was further examined as a function of reductase concentration. At lower ionic strength, there was a significant synergistic stimulation of EROD. This synergistic stimulation diminished with increasing reductase concentration, resulting in an additive response as reductase became saturating. Interestingly, at high ionic strength, the synergism of EROD in the mixed reconstituted system was not observed. In contrast, mixed reconstituted systems containing CYP2E1 and CYP2B4 did not provide evidence for the formation of these heteromeric P450-P450 complexes. The synergistic stimulation observed with the reductase-CYP1A2-CYP2E1 mixed reconstituted system is consistent with the formation of a CYP1A2-CYP2E1 complex. Taken together with the lack of a kinetically detectable interaction between CYP2B4 and CYP2E1, and the previously reported CYP1A2-CYP2B4 interaction, these results suggest that CYP1A2 may facilitate the formation of complexes with other P450 enzymes.     

Man8GlcNAc2糖基化的酿酒酵母菌株的构建

酿酒酵母糖蛋白的N-糖基化经过高尔基体的修饰后形成聚合度约150-200的甘露寡糖,高尔基体N-糖基化的糖基转移酶Mnn1p和Och1p在甘露寡糖的形成过程中起关键作用。通过同源重组置换敲除了酵母中的MNN1和OCH1基因阻断高尔基体N-糖基化修饰,分离纯化了mnn1 och1突变株中的N-糖蛋白,糖酰胺酶PNGaseF酶解释放的N-糖链经过2-氨基吡啶衍生后,利用HPLC和MALDITOF/MS结合的方法分析了突变株糖蛋白上的N-糖链。结果显示mnn1 och1突变株中的糖蛋白的N-糖链为结构单一的糖链,分子量为1794.66,推测为Man₈GlcNAc₂。     

The role of N-linked glycosylation in determining the surface expression,G protein interaction and effector coupling of the alpha (alpha) isoform of the human thromboxane A(2) receptor

In humans, thromboxane (TX) A(2) signals through two TXA(2) receptor (TP) isoforms, termed TPalpha and TPbeta, that diverge exclusively within the carboxyl terminal cytoplasmic domains. The amino terminal extracellular region of the TPs contains two highly conserved Asn (N)-linked glycosylation sites at Asn(4) and Asn(16). While it has been established that impairment of N-glycosylation of TPalpha significantly affects ligand binding/intracellular signalling, previous studies did not ascertain whether N-linked glycosylation was critical for ligand binding per se or whether it was required for the intracellular trafficking and the functional expression of TPalpha on the plasma membrane (PM). In the current study, we investigated the role of N-linked glycosylation in determining the functional expression of TPalpha, by assessment of its ligand binding, G protein coupling and intracellular signalling properties, correlating it with the level of antigenic TPalpha protein expressed on the PM and/or retained intracellularly. From our data, we conclude that N-glycosylation of either Asn(4) or Asn(16) is required and sufficient for expression of functionally active TPalpha on the PM while the fully non-glycosylated TPalpha(N4,N16-Q4,Q16) is almost completely retained within the endoplasmic reticulum (ER) and remains functionally inactive, failing to associate with its coupling G protein Galpha(q) and, in turn, failing to mediate phospholipase (PL) Cbeta activation.     

The sequence NPFXD defines a new class of endocytosis signal in Saccharomyces cerevisiae

The yeast membrane protein Kex2p uses a tyrosine-containing motif within the cytoplasmic domain for localization to a late Golgi compartment. Because Golgi membrane proteins mislocalized to the plasma membrane in yeast can undergo endocytosis, we examined whether the Golgi localization sequence or other sequences in the Kex2p cytoplasmic domain mediate endocytosis. To assess endocytic function, the Kex2p cytoplasmic domain was fused to an endocytosis-defective form of the alpha-factor receptor. Ste2p. Like intact Ste2p, the chimeric protein, Stex22p, undergoes rapid endocytosis that is dependent on clathrin and End3p. Uptake of Stex22p does not require the Kex2p Golgi localization motif. Instead, the sequence NPFSD, located 37 amino acids from the COOH terminus, is essential for Stex22p endocytosis. Internalization was abolished when the N, P, or F residues were converted to alanine and severely impaired upon conversion of D to A. NPFSD restored uptake when added to the COOH terminus of an endocytosis-defective Ste2p chimera lacking lysine-based endocytosis signals present in wild-type Ste2p. An NPF sequence is present in the cytoplasmic domain of the a- factor receptor, Ste3p. Mutation of this sequence prevented pheromone- stimulated endocytosis of a truncated form of Ste3p. Our results identify NPFSD as a clathrin-dependent endocytosis signal that is distinct from the aromatic amino acid-containing Golgi localization motif and lysine-based, ubiquitin-dependent endocytosis signals in yeast.     

Recognition of the carboxyl-terminal signal for GPI modification requires translocation of its hydrophobic domain across the ER membrane

A carboxyl-terminal hydrophobic domain is an essential component of the processed signal for attachment of the glycosyl-phosphatidylinositol (GPI) membrane anchor to proteins and it is linked to the site (omega) of GPI modification by a spacer domain. This study was designed to test the hypothesis that the hydrophobic domain interacts with the lipid bilayer of the endoplasmic reticulum (ER) membrane to optimally position the omega site for GPI modification. The hydrophobic domain of the GPI signal in the human folate receptor (FR) type alpha was substituted with the carboxyl-terminal segment of the low-density lipoprotein receptor (LDLR), including its membrane spanning region, without altering either the spacer or the omega site. The FR-alpha/LDLR chimera was not GPI modified but was attached to the plasma membrane by a polypeptide anchor. When the carboxyl-terminal half of the hydrophobic transmembrane polypeptide in the FR-alpha/LDLR chimera was altered by introduction of negatively charged (Asp) residues, or when the cytosolic domain in the chimera was deleted, the mutated proteins became GPI-anchored. On the other hand, attachment of a carboxyl-terminal segment of LDLR including the entire cytosolic domain to FR-alpha converted it into a transmembrane protein. The results indicate that in the FR-alpha/LDLR chimera the inability of the cellular machinery for GPI modification to recognize the hydrophobic domain is not due to the intrinsic nature of the peptide, but is rather due to the retention of the peptide within the lipid bilayer. It follows that the hydrophobic domain in the signal for GPI modification must traverse the ER membrane prior to recognition of the omega site by the GPI-protein transamidase. The results thus establish a critical topographical requirement for recognition of the GPI signal in the ER.