Analysis of the role of the EnvZ linker region in signal transduction using a chimeric Tar/EnvZ receptor protein,Tez1

Tez1 is a chimeric protein in which the periplasmic and transmembrane domains of Tar, a chemosensor, are fused to the cytoplasmic catalytic domain of EnvZ, an osmosensing histidine kinase, through the EnvZ linker. Unlike Taz1 (a similar hybrid with the Tar linker), Tez1 could not respond to Tar ligand, aspartate, whereas single Ala insertion at the transmembrane/linker junction, as seen in Tez1A1, restored the aspartate-regulatable phenotype. Analysis of the Ala insertion site requirement and the nature of the insertion residue on the phenotype of Tez1 indicated that a junction region between the transmembrane domain and the predicted helix I in the linker is critical to signal transduction. Random mutagenesis revealed that P185Q mutation in the Tez1 linker restored the aspartate-regulatable phenotype. Substitution mutations at Pro-185 further demonstrated that specific residues are required at this site for an aspartate response. None of the hybrid receptors constructed with different Tar/EnvZ fusion sites in the linker could respond to aspartate, suggesting that specific interactions between the two predicted helices in the linker are important for the linker function. In addition, a mutation (F220D) known to cause an OmpCc phenotype in EnvZ resulted in similar OmpCc phenotypes in both Tez1A1 and Tez1, indicating the importance of the predicted helix II in signal propagation. Together, we propose that the N-terminal junction region modulates the alignment between the two helices in the linker upon signal input. In turn helix II propagates the resultant conformational signal into the downstream catalytic domain of EnvZ to regulate its bifunctional enzymatic activities.     

The structural context of disease-causing mutations in gap junctions

Gap junctions form intercellular channels that mediate metabolic and electrical signaling between neighboring cells in a tissue. Lack of an atomic resolution structure of the gap junction has made it difficult to identify interactions that stabilize its transmembrane domain. Using a recently computed model of this domain, which specifies the locations of each amino acid, we postulated the existence of several interactions and tested them experimentally. We introduced mutations within the transmembrane domain of the gap junction-forming protein connexin that were previously implicated in genetic diseases and that apparently destabilized the gap junction, as evidenced here by the absence of the protein from the sites of cell-cell apposition. The model structure helped identify positions on adjacent helices where second-site mutations restored membrane localization, revealing possible interactions between residue pairs. We thus identified two putative salt bridges and one pair involved in packing interactions in which one disease-causing mutation suppressed the effects of another. These results seem to reveal some of the physical forces that underlie the structural stability of the gap junction transmembrane domain and suggest that abrogation of such interactions bring about some of the effects of disease-causing mutations.     

Interhelical hydrogen bonds in the CFTR membrane domain.

Critical mutations in the membrane-spanning domains of proteins cause many human diseases. We report the expression in Escherichia coli of helix-loop-helix segments of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel domain in milligram quantities. Analysis of gel migration patterns of these constructs, in conjunction with circular dichroism spectroscopy, demonstrate that a neutral-to-charged, CF-phenotypic point mutation of a hydrophobic residue (V232D) in the CFTR transmembrane (TM) helix 4 induces a hydrogen bond with neighboring wild type Gln 207 in TM helix 3. As an electrostatic crosslink within a hydrocarbon phase, such a hydrogen bond could alter the normal assembly and alignment of CFTR TM helices and/or impede their movement in response to substrate transport. Our results imply that membrane proteins may be vulnerable to loss of function through formation of membrane-buried interhelical hydrogen bonds by partnering of proximal polar side chains.     

The Gene Mutated in Variant Late-Infantile Neuronal Ceroid Lipofuscinosis (CLN6) and in nclf Mutant Mice Encodes a Novel Predicted Transmembrane Protein

The neuronal ceroid lipofuscinoses (NCLs) are a group of autosomal recessive neurodegenerative diseases characterized by the accumulation of autofluorescent lipopigment in various tissues and by progressive cell death in the brain and retina. The gene for variant late-infantile NCL (vLINCL), CLN6, was previously mapped to chromosome 15q21-23 and is predicted to be orthologous to the genes underlying NCL in nclf mice and in South Hampshire and Merino sheep. The gene underlying this disease has been identified with six different mutations found in affected patients and with a 1-bp insertion in the orthologous Cln6 gene in the nclf mouse. CLN6 encodes a novel 311-amino acid protein with seven predicted transmembrane domains, is conserved across vertebrates and has no homologies with proteins of known function. One vLINCL mutation, affecting a conserved amino acid residue within the predicted third hydrophilic loop of the protein, has been identified, suggesting that this domain may play an important functional role.     

High resolution NMR analysis of the seven transmembrane domains of a heptahelical receptor in organic-aqueous medium

The NMR properties of seven peptides representing the transmembrane domains of the alpha-factor receptor from Saccharomyces cerevisiae were examined in trifluoroethanol/water (4:1) at 10 to 55 degrees C. The parameters extracted indicated all peptides were helical in this membrane mimetic solvent. Using chemical shift indices as the criterion, helicity varied from 64 to 83%. The helical residues in the peptides corresponded to the region predicted to cross the hydrocarbon interior of the bilayer. A study of a truncated 25-residue peptide corresponding to domain 2 gave evidence that the helix extended all the way to the N-terminus of this peptide, indicating that sequence and not chain end effects are very important in helix termination for our model peptides. Both nuclear Overhauser effect spectroscopy (NOESY) connectivities and chemical shift indices revealed significant perturbations around prolyl residues in the helices formed by transmembrane domains 6 and 7. Molecular models of the transmembrane domains indicate that helices for domains 6 and 7 are severely kinked at these prolyl residues. The helix perturbation around proline 258 in transmembrane domain 6 correlates with mutations that cause phenotypic changes in this receptor.     

Membrane topology of the colicin E1 channel using genetically encoded fluorescence

The membrane topology of the colicin E1 channel domain was studied by fluorescence resonance energy transfer (FRET). The FRET involved a genetically encoded fluorescent amino acid (coumarin) as the donor and a selectively labeled cysteine residue tethered with DABMI (4-(dimethylamino)phenylazophenyl-4'-maleimide) as the FRET acceptor. The fluorescent coumarin residue was incorporated into the protein via an orthogonal tRNA/aminoacyl-tRNA synthetase pair that allowed selective incorporation into any site within the colicin channel domain. Each variant harbored a stop (TAG) mutation for coumarin incorporation and a cysteine (TGT) mutation for DABMI attachment. Six interhelical distances within helices 1-6 were determined using FRET analysis for both the soluble and membrane-bound states. The FRET data showed large changes in the interhelical distances among helices 3-6 upon membrane association providing new insight into the membrane-bound structure of the channel domain. In general, the coumarin-DABMI FRET interhelical efficiencies decreased upon membrane binding, building upon the umbrella model for the colicin channel. A tentative model for the closed state of the channel domain was developed based on current and previously published FRET data. The model suggests circular arrangement of helices 1-7 in a clockwise direction from the extracellular side and membrane interfacial association of helices 1, 6, 7, and 10 around the central transmembrane hairpin formed by helices 8 and 9.     

Syncytium-inducing mutations localize to two discrete regions within the cytoplasmic domain of herpes simplex virus type 1 glycoprotein B.

Herpes simplex virus type 1 glycoprotein B (gB) is essential for virus entry, an event involving fusion of the virus envelope with the cell surface membrane, and virus-induced cell-cell fusion, resulting in polykaryocyte, or syncytium, formation. The experiments described in this report employed a random mutagenesis strategy to develop a more complete genetic map of mutations resulting in the syn mutant phenotype. The results indicate that syn mutations occur within two essential and highly conserved hydrophilic, alpha-helical regions of the gB cytoplasmic domain. Region I is immediately proximal to the transmembrane domain and includes residues R796 to E816/817. Region II is localized centrally in the cytoplasmic domain and includes residues A855 and R858. Positively charged residues were particularly affected in both regions, suggesting that charge interactions may be required to suppress the syn mutant phenotype. No syn mutations were identified within the transmembrane domain. A virus containing a rate of entry (roe) mutation at residue A851, either within or immediately proximal to syn region II, was isolated. Since roe mutations have also been discovered in the external domain of gB, it appears likely that the external and cytoplasmic domains cooperate in virus penetration. Moreover, the observation that both roe and syn mutations occur in the cytoplasmic domain further suggests that gB functions in an analogous manner in both membrane fusion events. It might be predicted from these observations that membrane fusion involves transduction of a fusion signal along the gB molecule through the transmembrane domain. Communication between the external and cytoplasmic domain may thus be required for gB-mediated membrane fusion events.     

Cloning the mouse homolog of the human cystic fibrosis transmembrane conductance regulator gene.

The cystic fibrosis transmembrane conductance regulator is encoded by the gene known to be mutated in patients with cystic fibrosis. This paper reports the cloning and sequencing of cDNAs for the murine homolog of the human cystic fibrosis transmembrane conductance regulator gene. A clone that, by analogy to the human sequence, extends 3' from exon 9 to the poly(A) tail was isolated from a mouse lung cDNA library. cDNA clones containing exons 4 and 6b were also isolated and sequenced, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-9 were cloned by PCR from mouse RNA. The deduced mouse protein sequence is 78% identical to the human cystic fibrosis transmembrane regulator, with higher conservation in the transmembrane and nucleotide-binding domains. Amino acid sequences in which known cystic fibrosis missense mutations occur are conserved between man and mouse; in particular, the predicted mouse protein has a phenylalanine residue corresponding to that deleted in the most common human cystic fibrosis mutation (delta F508), which should allow the use of transgenic strategies to introduce this mutation in attempts to create a "cystic fibrosis mouse".     

Arabidopsis Acyl-CoA-Binding Protein ACBP2 Interacts With an Ethylene-Responsive Element-Binding Protein,AtEBP, via its Ankyrin Repeats

Cytosolic acyl-CoA-binding proteins (ACBP) bind long-chain acyl-CoAs and act as intracellular acyl-CoA transporters and maintain acyl-CoA pools. Arabidopsis thaliana ACBP2 shows conservation at the acyl-CoA-binding domain to cytosolic ACBPs but is distinct by the presence of an N-terminal transmembrane domain and C-terminal ankyrin repeats. The function of the acyl-CoA-binding domain in ACBP2 has been confirmed by site-directed mutagenesis and four conserved residues crucial for palmitoyl-CoA binding have been identified. Results from ACBP2:GFP fusions transiently expressed in onion epidermal cells have demonstrated that the transmembrane domain functions in plasma membrane targeting, suggesting that ACBP2 transfers acyl-CoA esters to this membrane. In this study, we investigated the significance of its ankyrin repeats in mediating protein-protein interactions by yeast two-hybrid analysis and in vitro protein-binding assays; we showed that ACBP2 interacts with the A. thaliana ethylene-responsive element-binding protein AtEBP via its ankyrin repeats. This interaction was lacking in yeast two-hybrid analysis upon removal of the ankyrin repeats. When the subcellular localizations of ACBP2 and AtEBP were further investigated using autofluorescent protein fusions in transient expression by agroinfiltration of tobacco leaves, the DsRed:ACBP2 fusion protein was localized to the plasma membrane while the GFP:AtEBP fusion protein was targeted to the nucleus and plasma membrane. Co-expression of DsRed:ACBP2 and GFP:AtEBP showed a common localization of both proteins at the plasma membrane, suggesting that ACBP2 likely interacts with AtEBP at the plasma membrane.     

Polar residues in membrane domains of proteins: molecular basis for helix-helix association in a mutant CFTR transmembrane segment

Polar side chains constitute over 20% of residues in the transmembrane (TM) helices of membrane proteins, where they may serve as hydrogen bond interaction sites for phenotypic polar mutations that arise in membrane protein-related diseases. To systematically explore the structural consequences of H-bonds between TM helices, we focused on TM4 of the cystic fibrosis conductance regulator (CFTR) and its cystic fibrosis- (CF-) phenotypic mutation, V232D, as a model system. Synthetic peptides corresponding to wild-type (TM4-wt) (residues 219-242: LQASAFCGLGFLIVLALFQAGLGR) and mutant (TM4-V232D) sequences both adopt helical structures in SDS micelles and display dimer bands on SDS-PAGE arising from disulfide bond formation via wild-type residue Cys-225. However, the TM4-V232D peptide additionally forms a ladder of noncovalent oligomers, including tetramers, hexamers, and octamers, mediated by a hydrogen bond network involving Asp-Gln side chain-side chain interactions. Ala-scanning mutagenesis of the TM4 sequence indicated that ladder formation minimally required the simultaneous presence of the Cys-225, Asp-232, and Gln-237 residues. As random hydrophobic sequences containing these three residues at TM4 equivalent positions did not oligomerize, specific van der Waals packing interactions between helix side chains were also shown to play a crucial role. Overall, the results suggest that polar mutations in membrane domains, in conjunction with critically positioned polar partner residues, potentially constitute a source of aberrant helix interactions that could contribute to loss of function when they arise in protein transmembrane domains.     

Interaction between transmembrane domains five and six of the alpha -factor receptor

The alpha-factor pheromone receptor (STE2) activates a G protein signal pathway that induces conjugation of the yeast Saccharomyces cerevisiae. Previous studies implicated the third intracellular loop of this receptor in G protein activation. Therefore, the roles of transmembrane domains five and six (TMD5 and -6) that bracket the third intracellular loop were analyzed by scanning mutagenesis in which each residue was substituted with cysteine. Out of 42 mutants examined, four constitutive mutants and two strong loss-of-function mutants were identified. Double mutants combining Cys substitutions in TMD5 and TMD6 gave a broader range of phenotypes. Interestingly, a V223C mutation in TMD5 caused constitutive activity when combined with the L247C, L248C, or S251C mutations in TMD6. Also, the L226C mutation in TMD5 caused constitutive activity when combined with either the M250C or S251C mutations in TMD6. The residues affected by these mutations are predicted to fall on one side of their respective helices, suggesting that they may interact. In support of this, cysteines substituted at position 223 in TMD5 and position 247 in TMD6 formed a disulfide bond, providing the first direct evidence of an interaction between these transmembrane domains in the alpha-factor receptor. Altogether, these results identify an important region of interaction between conserved hydrophobic regions at the base of TMD5 and TMD6 that is required for the proper regulation of receptor signaling.     

Requirement of Calcium Binding,Myristoylation, and Protein-Protein Interaction for the Copine BON1 Function in Arabidopsis

Copines are highly conserved proteins with lipid-binding activities found in animals, plants, and protists. They contain two calcium-dependent phospholipid binding C2 domains at the amino terminus and a VWA domain at the carboxyl terminus. The biological roles of most copines are not understood and the biochemical properties required for their functions are largely unknown. The Arabidopsis copine gene BON1/CPN1 is a negative regulator of cell death and defense responses. Here we probed the potential biochemical activities of BON1 through mutagenic studies. We found that mutations of aspartates in the C2 domains did not alter plasma membrane localization but compromised BON1 activity. Mutation at putative myristoylation residue glycine 2 altered plasma membrane localization of BON1 and rendered BON1 inactive. Mass spectrometry analysis of BON1 further suggests that the N-peptide of BON1 is modified. Furthermore, mutations that affect the interaction between BON1 and its functional partner BAP1 abolished BON1 function. This analysis reveals an unanticipated regulation of copine protein localization and function by calcium and lipid modification and suggests an important role in protein-protein interaction for the VWA domain of copines.     

Transmembrane segment 12 of the Glut1 glucose transporter is an outer helix and is not directly involved in the transport mechanism

A model has been proposed for the exofacial configuration of the Glut1 glucose transporter in which eight transmembrane domains form an inner helical bundle stabilized by four outer helices. The role of transmembrane segment 12, predicted to be an outer helix in this hypothetical model, was examined by cysteine-scanning mutagenesis and the substituted cysteine accessibility method using the membrane-impermeant, sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). A previously characterized functional cysteine-less Glut1 molecule was used to produce 21 Glut1 point mutants by changing each residue along helix 12 to a cysteine residue. These mutants were then expressed in Xenopus oocytes, and their protein levels, functional activities, and sensitivities to pCMBS were determined. Strikingly, in contrast to all nine other predicted Glut1 transmembrane helices that have been previously examined by this method, none of the 21 helix 12 single-cysteine mutants exhibited significant inhibition of specific transport activity. Also unlike most other Glut1 transmembrane domains in which solvent-accessible residues lie along a single face of the helix, mutations in five consecutive residues predicted to lie close to the exofacial face of the membrane resulted in sensitivity to pCMBS-induced transport inhibition. These results suggest that helix 12 plays a passive stabilizing role in the structure of Glut1 and is not directly involved in the transport mechanism. Additionally, the pCMBS data indicate that the predicted exoplasmic end of helix 12 is completely exposed to the external solvent when the transporter is in its exofacial configuration.     

Impact of the deltaF508 mutation in first nucleotide-binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure

Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.     

Insertion of an arginine residue into the transmembrane segments corrects protein misfolding

Deletion of Phe-508 (DeltaF508) in cystic fibrosis transmembrane conductance regulator causes cystic fibrosis because of misfolding of the protein. P-glycoprotein (P-gp) containing the equivalent mutation (DeltaY490) is also misfolded but can be rescued with drug substrates. Whether rescue is due to direct binding of drug substrate to the transmembrane (TM) segments or to indirect effects on cellular protein folding pathways is still controversial. P-gp-drug substrate interactions likely involve hydrogen bonds. If the mechanism of drug rescue involves changes to TM packing then we should be able to identify suppressor mutations in the TM segments that can mimic the drug rescue effects. We predicted that an arginine residue in the TM segments predicted to line the drug-binding pocket of P-gp (I306(TM5) or F343(TM6)) might suppress DeltaY490 P-gp protein misfolding because it has the highest propensity to form hydrogen bonds. We show that R306(TM5) or R343(TM6) increased the relative amount of mature DeltaY490 P-gp by 6-fold. Most other changes to Ile-306 or Phe-343 did not enhance maturation of DeltaY490 P-gp. The I306R mutant also promoted maturation of misprocessed mutants that had mutations in the second nucleotide-binding domain (L1260A), the cytoplasmic loops (G251V, F804A), the linker region (P709A), or in TM segments (G300V, G722A). These results show that arginine residues in the TM domains can mimic the drug rescue effects and are effective suppressor mutations for processing mutations located throughout the molecule.     

Thermostability of membrane protein helix-helix interaction elucidated by statistical analysis

A prerequisite for the survival of (micro)organisms at high temperatures is an adaptation of protein stability to extreme environmental conditions. In contrast to soluble proteins, where many factors have already been identified, the mechanisms by which the thermostability of membrane proteins is enhanced are almost unknown. The hydrophobic membrane environment constrains possible stabilizing factors for transmembrane domains, so that a difference might be expected between soluble and membrane proteins. Here we present sequence analysis of predicted transmembrane helices of the genomes from eight thermophilic and 12 mesophilic organisms. A comparison of the amino acid compositions indicates that more polar residues can be found in the transmembrane helices of thermophilic organisms. Particularly, the amino acids aspartic acid and glutamic acid replace the corresponding amides. Cysteine residues are found to be significantly decreased by about 70% in thermophilic membrane domains suggesting a non-specific function of most cysteine residues in transmembrane domains of mesophilic organisms. By a pair-motif analysis of the two sets of transmembrane helices, we found that the small residues glycine and serine contribute more to transmembrane helix-helix interactions in thermophilic organisms. This may result in a tighter packing of the helices allowing more hydrogen bond formation.     

N-terminal motifs in some plant disease resistance proteins function in membrane attachment and contribute to disease resistance

To investigate the role of N-terminal domains of plant disease resistance proteins in membrane targeting, the N termini of a number of Arabidopsis and flax disease resistance proteins were fused to green fluorescent protein (GFP) and the fusion proteins localized in planta using confocal microscopy. The N termini of the Arabidopsis RPP1-WsB and RPS5 resistance proteins and the PBS1 protein, which is required for RPS5 resistance, targeted GFP to the plasma membrane, and mutation of predicted myristoylation and potential palmitoylation sites resulted in a shift to nucleocytosolic localization. The N-terminal domain of the membrane-attached Arabidopsis RPS2 resistance protein was targeted incompletely to the plasma membrane. In contrast, the N-terminal domains of the Arabidopsis RPP1-WsA and flax L6 and M resistance proteins, which carry predicted signal anchors, were targeted to the endomembrane system, RPP1-WsA to the endoplasmic reticulum and the Golgi apparatus, L6 to the Golgi apparatus, and M to the tonoplast. Full-length L6 was also targeted to the Golgi apparatus. Site-directed mutagenesis of six nonconserved amino acid residues in the signal anchor domains of L6 and M was used to change the localization of the L6 N-terminal fusion protein to that of M and vice versa, showing that these residues control the targeting specificity of the signal anchor. Replacement of the signal anchor domain of L6 by that of M did not affect L6 protein accumulation or resistance against flax rust expressing AvrL567 but removal of the signal anchor domain reduced L6 protein accumulation and L6 resistance, suggesting that membrane attachment is required to stabilize the L6 protein.     

Structure prediction of helical transmembrane proteins at two length scales

As the first step toward a multi-scale, hierarchical computational approach for membrane protein structure prediction, the packing of transmembrane helices was modeled at the residue and atom levels, respectively. For predictions at the residue level, the helix-helix and helix-membrane interactions were described by a set of knowledge-based energy functions. For predictions at the atom level, CHARMM19 force field was used. To facilitate the system to overcome energy barriers, the Wang-Landau method was employed, where a random walk is performed in the energy space with a uniform probability. Native-like structures were predicted at both levels for two model systems, each of which consists of two transmembrane helices. Interestingly, consistent results were obtained from simulations at the residue and atom levels for the same system, strongly suggesting the feasibility of a hierarchical approach for membrane protein structure predictions.     

Membrane anchoring domain of herpes simplex virus glycoprotein gB is sufficient for nuclear envelope localization.

We have used the glycoprotein gB of herpes simplex virus type 1 (gB-1), which buds from the inner nuclear membrane, as a model protein to study localization of membrane proteins in the nuclear envelope. To determine whether specific domains of gB-1 glycoprotein are involved in localization in the nuclear envelope, we have used deletion mutants of gB-1 protein as well as chimeric proteins constructed by replacing the domains of the cell surface glycoprotein G of vesicular stomatitis virus with the corresponding domains of gB. Mutant and chimeric proteins expressed in COS cells were localized by immunoelectron microscopy. A chimeric protein (gB-G) containing the ectodomain of gB and the transmembrane and cytoplasmic domains of G did not localize in the nuclear envelope. When the ectodomain of G was fused to the transmembrane and cytoplasmic domains of gB, however, the resulting chimeric protein (G-gB) was localized in the nuclear envelope. Substitution of the transmembrane domain of G with the 69 hydrophobic amino acids containing the membrane anchoring domain of gB allowed the hybrid protein (G-tmgB) to be localized in the nuclear envelope, suggesting that residues 721 to 795 of gB can promote retention of proteins in the nuclear envelope. Deletion mutations in the hydrophobic region further showed that a transmembrane segment of 21 hydrophobic amino acids, residues 774 to 795 of gB, was sufficient for localization in the nuclear envelope. Since wild-type gB and the mutant and chimeric proteins that were localized in the nuclear envelope were also retained in the endoplasmic reticulum, the membrane spanning segment of gB could also influence retention in the endoplasmic reticulum.