Molecular Cloning of Human Plasma Membrane Phospholipid Scramblase

The rapid movement of phospholipids (PL) between plasma membrane leaflets in response to increased intracellular Ca2+ is thought to play a key role in expression of platelet procoagulant activity and in clearance of injured or apoptotic cells. We recently reported isolation of a ∼37-kDa protein in erythrocyte membrane that mediates Ca2+-dependent movement of PL between membrane leaflets, similar to that observed upon elevation of Ca2+ in the cytosol (Bassé, F., Stout, J. G., Sims, P. J., and Wiedmer, T. (1996) J. Biol. Chem.271, 17205–17210). Based on internal peptide sequence obtained from this protein, a 1,445-base pair cDNA was cloned from a K-562 cDNA library. The deduced “PL scramblase” protein is a proline-rich, type II plasma membrane protein with a single transmembrane segment near the C terminus. Antibody against the deduced C-terminal peptide was found to precipitate the ∼37-kDa red blood cell protein and absorb PL scramblase activity, confirming the identity of the cloned cDNA to erythrocyte PL scramblase. Ca2+-dependent PL scramblase activity was also demonstrated in recombinant protein expressed from plasmid containing the cDNA. Quantitative immunoblotting revealed an approximately 10-fold higher abundance of PL scramblase in platelet (∼104 molecules/cell) than in erythrocyte (∼103 molecules/cell), consistent with apparent increased PL scramblase activity of the platelet plasma membrane. PL scramblase mRNA was found in a variety of hematologic and nonhematologic cells and tissues, suggesting that this protein functions in all cells.

The rapid movement of phospholipids (PL) between plasma membrane leaflets in response to increased intracellular Ca 2؉ is thought to play a key role in expression of platelet procoagulant activity and in clearance of injured or apoptotic cells. We recently reported isolation of a ϳ37-kDa protein in erythrocyte membrane that mediates Ca 2؉ -dependent movement of PL between membrane leaflets, similar to that observed upon elevation of Ca 2؉ in the cytosol (Bassé , F., Stout, J. G., Sims, P. J., and Wiedmer, T. (1996) J. Biol. Chem. 271, 17205-17210). Based on internal peptide sequence obtained from this protein, a 1,445-base pair cDNA was cloned from a K-562 cDNA library. The deduced ''PL scramblase'' protein is a proline-rich, type II plasma membrane protein with a single transmembrane segment near the C terminus. Antibody against the deduced Cterminal peptide was found to precipitate the ϳ37-kDa red blood cell protein and absorb PL scramblase activity, confirming the identity of the cloned cDNA to erythrocyte PL scramblase. Ca 2؉ -dependent PL scramblase activity was also demonstrated in recombinant protein expressed from plasmid containing the cDNA. Quantitative immunoblotting revealed an approximately 10-fold higher abundance of PL scramblase in platelet (ϳ10 4 molecules/cell) than in erythrocyte (ϳ10 3 molecules/ cell), consistent with apparent increased PL scramblase activity of the platelet plasma membrane. PL scramblase mRNA was found in a variety of hematologic and nonhematologic cells and tissues, suggesting that this protein functions in all cells.
The plasma membrane phospholipids (PL) 1 are normally asymmetrically distributed, with phosphatidylcholine (PC) and sphingomyelin located primarily in the outer leaflet, and the aminophospholipids, phosphatidylserine (PS) and phosphati-dylethanolamine restricted to the cytoplasmic leaflet (1,2). An increase in intracellular Ca 2ϩ due to either cell activation, cell injury, or apoptosis causes a rapid bidirectional movement of the plasma membrane PL between leaflets, resulting in exposure of PS and phosphatidylethanolamine at the cell surface (1,(3)(4)(5). This exposure of the plasma membrane aminophospholipids has been shown to promote assembly and activation of several key enzymes of the coagulation and complement systems, as well as to accelerate the clearance of injured or apoptotic cells by the reticuloendothelial system, suggesting that Ca 2ϩ -induced remodeling of plasma membrane PL is central to both vascular hemostatic and cellular clearance mechanisms (1, 6 -9).
We recently reported isolation of a ϳ37-kDa integral membrane protein from human erythrocytes that when reconstituted into liposomes mediated a Ca 2ϩ -dependent, bidirectional scrambling of PL between membrane leaflets mimicking the action of Ca 2ϩ at the endofacial surface of the erythrocyte membrane (10,11). Evidence for protein(s) in platelet that mediates a similar "PL scramblase" function when incorporated into liposomes has also been reported (12). Here we report the cDNA cloning and deduced structure of the PL scramblase from human erythrocyte and show evidence that this same protein is expressed in human platelet and various other cell lines and tissues where plasma membrane PL scramblase activity has been observed.
PL Scramblase Isolation and Amino Acid Sequencing-PL scramblase was purified as described previously (10,11), with the following modifications. The active fraction eluting from Mono S was concentrated and exchanged into 150 mM NaCl, 20 mM Tris, 0.1 mM EGTA, 0.1% Nonidet P-40, pH 7.4, and absorbed against 5 ml of wheat germ agglutinin-Sepharose to remove trace contaminating glycophorins. The breakthrough material was concentrated and exchanged into 20 mM Tris, 0.1 mM EGTA, 0.02% Nonidet P-40, pH 7.4, and subjected to SDS-PAGE under reducing conditions in a 10% NuPAGE gel (Novex, San Diego, CA). The band at ϳ37 kDa was visualized with 0.1% Brilliant Blue R-250 and excised for amino acid analysis and sequencing (University of Michigan Protein and Carbohydrate Structure Facility). 450 pmol of this protein was subjected to in situ cleavage with 10 mg/ml CNBr in 70% formic acid, the cleaved peptides were extracted into 60% acetonitrile, 10% trifluoroacetic acid, dried in a speed vacuum, and * This work was supported in part by Grant R01 HL36946 from the NHLBI, National Institutes of Health (to P. J. S. and T. W.) and a Grant-In-Aid from the American Heart Association (to T. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Isolation of PL Scramblase cDNA by Plaque Hybridization-The 568-bp insert of EST clone gb AA143025 was labeled with [␣-32 P]dCTP by Random Primed DNA Labeling Kit (Boehringer Mannheim) and used to screen a cDNA library derived from human erythroleukemic cell line K-562 in gt11 (CLONTECH). Escherichia coli strain Y1090r was transformed by K-562 cDNA library (4.86 ϫ 10 5 pfu) and poured on 27 agarose plates (15-cm diameter, 18,000 plaque-forming unit/plate). Plaques were lifted onto Hybond-N Nylon membranes (Amersham Corp.). After UV-cross-linking and prehybridization in a solution composed of 5 ϫ Denhardt, 5 ϫ SSC, 1% SDS, and 200 g/ml herring sperm DNA for 3 h at 68°C, the membranes were hybridized in the same solution containing 5 ng/ml 32 P-labeled probe for 16 h at 68°C. The membranes were washed once with 1 ϫ SSC, 0.1% SDS, then three times with 0.2 ϫ SSC, 0.1% SDS for 20 min at 65°C, and exposed to x-ray film. Secondary plaque lifts and hybridization were carried out on 32 strongly positive plaques at a density of 50 -100 plaques/plate. Single positive and well isolated plaques were picked and amplified. The length of cDNA insert was examined by PCR with gt11 reverse and forward primers. Six clones with cDNA inserts of Ͼ1.4 kilobase pairs were selected for DNA sequencing.
DNA Sequencing-DNA was sequenced on an Applied Biosystems DNA Sequencer model 373 Stretch using PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit (Perkin-Elmer) and combinations of vector and insert sequence primers.
Cloning of PL Scramblase into pMAL-C2 Expression Vector-To express PL scramblase as a fusion protein with maltose binding protein (MBP), cDNA encoding PL scramblase was cloned into pMAL-C2 (New England BioLabs). PCR was performed on a full-length clone using the primers 5Ј-TCAGAATTCGGATCCATGGACAAACAAAACTCACAGAT-G-3Ј with an EcoRI site before the ATG start codon and 5Ј-GCTTGCC-TGCAGGTCGACCTACCACACTCCTGATTTTTGTTCC-3Ј with a SalI site after the stop codon. KlenTaq polymerase (CLONTECH) was used to ensure high fidelity amplification. The PCR product was digested with EcoRI and SalI and isolated by electrophoresis on 1% low melting agarose gel and purification with Wizard kit (Promega). The amplified cDNA was cloned into pMAL-C2 vector digested with EcoRI and SalI, immediately 3Ј of MBP. This construct was amplified in E. coli strain TB1, and the sequence of the cDNA insert of plasmids from single colonies was confirmed.
Expression and Purification of PL Scramblase-MBP Fusion Protein-10 ml of E. coli TB1 transformed with scramblase cDNA-pMAL-C2 were used to inoculate 1 liter of rich LB containing 2 mg/ml glucose, 100 g/ml ampicillin, and the bacteria were allowed to grow for about 4 h at 37°C. When A 600 reached ϳ0.5, isopropyl-␤-D-thiogalactopyranoside was added to a final concentration of 0.3 mM. After 2 h of incubation at 37°C, the cells were centrifuged at 4000 ϫ g for 20 min. The cell pellet was suspended in 50 ml of 20 mM Tris, 200 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride (column buffer), and subjected to a freeze/thaw cycle. After sonication (3 ϫ 30 s on ice) and centrifugation at 43,000 ϫ g for 1 h, the supernatant was applied to 10 ml of amylose resin. The column was washed with 20 volumes of column buffer, and the scramblase-MBP fusion protein eluted with the same buffer containing 10 mM maltose. Digestion of MBP-PL scramblase protein with factor Xa was routinely performed at 1/100 (w/w) ratio of enzyme and monitored by SDS-PAGE. In addition to MBP, the product of this digest is the PL scramblase translation product containing the N-terminal extension Ile-Ser-Glu-Phe-Gly-Phe (codons Ϫ6 to Ϫ1).
Reconstitution of PL Scramblase or Scramblase Fusion Protein into Proteoliposomes-Reconstitution into proteoliposomes was performed essentially as described previously (10,11). Briefly, a mixture of PC and PS (9:1 molar ratio) was dried under a stream of nitrogen and resuspended in 100 mM Tris, 100 mM KCl, 0.1 mM EGTA, pH 7.4 (Tris buffer). Protein samples to be reconstituted were added to the liposomes at a final lipid concentration of 4 mg/ml in the presence of 60 mM OG and dialyzed overnight at 4°C against 200 volumes of Tris buffer containing 1 g/liter Bio-Beads SM-2. To liberate PL scramblase from MBP, the proteoliposomes were incubated for 3 h at room temperature in the presence of 1/40 (w/w) factor Xa. The digestion was terminated by the addition of 100 M Glu-Gly-Arg chloromethyl ketone. Completeness of the digest was monitored by SDS-PAGE. Following dialysis, the proteoliposomes were labeled in the outer leaflet by the addition of 0.25 mol % fluorescent NBD-PC (in dimethyl sulfoxide; final solvent concentration, 0.25%).
PL Scramblase Activity-PL Scramblase activity was measured as described previously (10,11). Routinely, proteoliposomes labeled with NBD-PC were incubated for 2 h at 37°C in Tris buffer in the presence or the absence of 2 mM CaCl 2 . Proteoliposomes were diluted 25-fold in Tris buffer containing 4 mM EGTA and transferred to a stirred fluorescence cuvette at 23°C. Initial fluorescence was recorded (SLM Aminco 8000 spectrofluorimeter; excitation, 470 nm; emission, 532 nm), 20 mM dithionite was added, and the fluorescence was continuously monitored for a total of 120 s. The difference in nonquenchable fluorescence observed in the presence versus the absence of CaCl 2 was attributed to Ca 2ϩ -induced change in NBD-PC located in the outer leaflet (10,11,13). Ionized [Ca 2ϩ ] was calculated using FreeCal version 4.0 software (generously provided by Dr. Lawrence F. Brass, University of Pennsylvania, Philadelphia, PA).
Antibody against PL Scramblase C-terminal Peptide-The peptide CESTGSQEQKSGVW, corresponding to amino acids 306 -318 of the predicted open reading frame of PL scramblase with an added Nterminal cysteine, was synthesized and conjugated to keyhole limpet hemocyanin (Protein Core Facility, Blood Research Institute). Antiserum to this protein was raised in rabbit (Cocalico Biologicals, Inc.), and the IgG fraction was isolated on protein A-Sepharose-CL4B (Sigma). Peptide-specific antibody was isolated by affinity chromatography on UltraLink Iodoacetyl beads (Pierce) to which peptide CES-TGSQEQKSGVW was conjugated. This affinity-purified antibody (anti-306 -318) was used for immunoprecipitation and Western blotting of PL scramblase (see ''Results and Discussion'').
Immunoprecipitation of PL Scramblase-PL scramblase purified from human erythrocytes was 125 I-labeled with Iodogen (Pierce), free iodide removed by gel filtration, and the protein was incubated (4°C, overnight) with either anti-306 -318,or an identical quantity of preimmune rabbit IgG (1 mg/ml in 150 mM NaCl, 10 mM MOPS, 50 mM OG, pH 7.4) or no IgG as control. The IgG was precipitated with protein A-Sepharose and washed exhaustively, and protein bands were resolved by 8 -25% SDS-PAGE (Phast System, Pharmacia Biotech Inc.) under reducing conditions. Radioactive bands were visualized by autoradiography. To determine whether antibody to this peptide specifically removed the functional activity associated with the purified erythrocyte PL scramblase protein, the supernatant fractions remaining after immunoprecipitation were reconstituted in liposomes for activity measurements, performed as described above. For these experiments, unlabeled erythrocyte PL scramblase substituted for the 125 I-labeled protein.
Western Blot Analysis-2 ϫ 10 8 washed platelets, 2 ϫ 10 8 erythrocyte ghost membranes, 0.9 pmol of purified recombinant PL scramblase (obtained by factor Xa digest of the PL scramblase-MBP fusion protein), and 0.3 pmol of PL scramblase purified from human erythrocyte were each denatured by boiling in 40 l of sample buffer containing 10% SDS, 4%␤-mercaptoethanol, and 1 mM EDTA, and protein bands were resolved by SDS-PAGE. After transfer to nitrocellulose, the blocked membrane was incubated with 1 g/ml of anti-306 -318, and the blot was developed with horseradish preoxidase-conjugated goat anti-rabbit IgG (Sigma) using Chemiluminescence Reagent (DuPont).
Protein Concentrations-Protein concentrations were estimated based upon optical density at 280 nm, using extinction coefficients (M Ϫ1 cm Ϫ1 ) of 39,000 (PL scramblase), 64,500 (MBP), and 105,000 (PL scramblase-MBP fusion). PL scramblase contained in human platelet and erythrocyte membranes was estimated by quantitative immunoblotting of the detergent extracts, with reference to known quantities of purified MBP-PL scramblase fusion protein.
Northern Blot Analysis-Human multiple tissue Northern blot and human cancer cell line multiple tissue Northern blot membranes were obtained from CLONTECH. The blots were prehybridized with Ex-pressHyb (CLONTECH) at 68°C for 30 min and hybridized with Ex-pressHyb containing 5 ng/ml 32 P-labeled PL scramblase cDNA probe at 68°C for 1 h, then washed, and exposed to x-ray film. After development, the blots were stripped and hybridized with 32 P-labeled ␤-actin cDNA probe using identical conditions.

RESULTS AND DISCUSSION
Cloning of PL Scramblase cDNA-PL scramblase was purified from human erythrocyte membranes and cleaved with cyanogen bromide, and Edman degradation was performed on a 12-kDa peptide fragment to obtain 32 residues of peptide sequence (Fig. 1, underlined sequence). This peptide sequence, plus the anticipated methionine residue N-terminal to the predicted site of cyanogen bromide cleavage, was identified in the translation product of a 568-bp EST clone deposited in Gen-Bank by the I.M.A.G.E. Consortium (clone identification number 505141). No other significant matches to this sequence were identified in any protein data base. The EST clone was used to screen a human K-562 leukemic cell cDNA library. Of 32 positive clones identified by plaque hybridization, six clones were sequenced yielding 1445 bp of cDNA (Fig. 1). The open reading frame encodes a protein without a signal sequence that contains 318 residues with a calculated mass of 35.1 kDa, a theoretical pI of 4.8, and a single predicted transmembrane helix near the C terminus (residues Ala 291 -Gly 309 ), in good agreement with the physical properties observed for the ϳ37-kDa protein band we tentatively identified as PL scramblase in human erythrocyte membrane (10). Whereas the deduced protein sequence is notable for its high proline (12%) content, homology searching failed to reveal significant concensus to identifiable protein family or domain structures, with the exception of a single potential protein kinase C phosphorylation site (Thr 161 ). The possibility that PL scramblase function is mediated by a phosphoprotein has previously been suggested based on an observed decrease in PL scrambling activity in erythrocytes depleted of ATP (14).
Analysis of the cDNA-derived protein sequence (Tmpred program, ISREC server, University of Lausanne, Epalinges, Switzerland) revealed a strongly preferred (p Ͻ 0.01) inside-to-outside orientation of the predicted 19-residue transmembrane helix, consistent with a type II plasma membrane protein. Most of the polypeptide (residues 1-290) thereby extends from the cytoplasmic membrane leaflet, leaving a short exoplasmic tail (residues 310 -318). The predicted orientation of this protein is consistent with the anticipated topology of PL scramblase in the erythrocyte membrane, where lipid-mobilizing function is responsive to [Ca 2ϩ ] only at the endofacial surface of the membrane (3,5,10,11,15,16).
To confirm that the cDNA we cloned from the K-562 cDNA library actually encodes the same protein purified as PL scramblase from human erythrocyte membrane, we raised a rabbit antibody against the deduced C terminus predicted from the open reading frame of the cloned cDNA (codons 306 -318). As shown in Fig. 2, this antibody precipitated the ϳ37-kDa red cell protein we tentatively identified as PL scramblase and also absorbed the functional activity detected in this isolated erythrocyte membrane protein fraction. As also evident from Fig. 2 (inset), we often observed the partial proteolysis of 37-kDa PL scramblase to a polypeptide of ϳ30 kDa. The apparent susceptibility of this protein to proteolytic degradation may account for the reported rapid loss of activity observed in earlier attempts to purify PL scramblase from platelet (12).
Expression and Membrane Reconstitution of Recombinant PL Scramblase-Recombinant PL scramblase was expressed in E. coli as fusion protein with MBP, purified by amylose affinity chromatography, and incorporated into PC/PS liposomes for assay of PL scramblase activity. When incorporated into liposomes, the recombinant protein mediated a Ca 2ϩ -dependent transbilayer movement of NBD-PC mimicking the activity of PL scramblase isolated from erythrocyte. PL scramblase activity was observed both for the chimeric MBP-PL scramblase fusion protein (not shown) and for recombinant PL scramblase liberated from MBP through proteolytic digestion with factor Xa (Fig. 3). By contrast, no such activity was observed for control protein consisting of the pMAL-C2 translation product MBP lacking the PL scramblase cDNA insert. The specific PL mobilizing activity of recombinant PL scramblase expressed and purified from E. coli was approximately 50% of that observed for the endogenous protein purified from the erythrocyte membrane, which is likely due to incomplete folding of the recombinant protein. Half-maximal [Ca 2ϩ ] required for activation was approximately 100 -200 M for recombinant protein purified from E. coli versus ϳ40 M for the erythrocyte-derived protein, raising the possibility that altered folding or an unknown post-translational modification in mammalian cells affects the putative Ca 2ϩ binding site (10,11). In addition to activation by Ca 2ϩ , the transbilayer migration of PL in erythrocytes is accelerated upon acidification of the inside leaflet to pH Ͻ 6.0 (in absence of Ca 2ϩ ), a response that is also observed in proteoliposomes containing PL scramblase purified from erythrocyte membranes (11). A similar acid-dependent activation of PL mobilizing function was also exhibited by proteoliposomes incorporating recombinant PL scramblase purified from E. coli (not shown).
Platelet PL Scramblase-In addition to the presumed role of PL scramblase in PS exposure following cell injury and upon repeated sickling of SS hemoglobin red cells, the capacity of activated platelets to rapidly mobilize aminophospholipids across the plasma membrane is thought to play a central role in the initiation of thrombin generation required for plasma clotting (17). Whereas incubation with Ca 2ϩ ionophore causes a marked acceleration in transbilayer movement of plasma membrane PL in both platelets and erythrocytes, the apparent rate of transbilayer PL migration in platelet exceeds that in erythrocyte by approximately 10-fold, implying either a higher abundance of PL scramblase or the action of another component in platelet with enhanced PL scrambling function (18,19). Zwaal and associates recently reported evidence for the existence of protein(s) in platelet with functional properties similar to that of PL scramblase we isolated from erythrocyte (10 -12). To determine whether the protein we now identify in the erythrocyte membrane is also found in platelets, we probed platelets with antibody against PL scramblase residues 306 -318. As shown in Fig. 4, this antibody blotted a single protein in platelet with similar mobility to the ϳ37-kDa PL scramblase in erythrocyte. Based on quantitative immunoblotting with anti-306 -318, we estimate approximately 10 4 molecules/cell in platelet versus 10 3 molecules/cell in erythrocyte, consistent with the increased PL scramblase activity and procoagulant function observed for human platelets versus erythrocytes.
Tissue Distribution-In addition to platelet and red blood cell, PL scramblase activity has been observed in many other cells, and this Ca 2ϩ -induced response is thought to be central to the rapid movement of PS and phosphatidylethanolamine from inner plasma membrane leaflet to the surface of perturbed endothelium and a variety of injured and apoptotic cells (17). The resulting exposure of PS at the cell surface is thought to play a key role in removal of such cells by the reticuloendothelial system, in addition to activation of both the plasma complement and coagulation systems (8,9,17). Whereas the molecular mechanism(s) in each circumstance remains unre- FIG. 3. Activity assay of recombinant PL scramblase. Purified PL scramblase-MBP fusion protein (0 -43 ϫ 10 Ϫ11 mol; abscissa) was reconstituted into liposomes (1 mol of total PL), and MBP was proteolytically removed by incubation with factor Xa in presence of 0.1 mM EGTA. After digest to release MBP, the proteoliposomes were recovered for determination of PL scramblase activity, measured in the absence (E) or the presence (q) of 2 mM CaCl 2 as described under "Experimental Procedures." Data are corrected for nonspecific transbilayer migration of NBD-PC probe in identically matched control liposomes containing either MBP or no added protein (Ͻ2% NBD-PC sequestered; not shown). Error bars denote the means Ϯ SD (n ϭ 3). Data of single experiment are shown, representative of two so performed. Similar results were also obtained for proteoliposomes containing intact PL scramblase-MBP fusion protein, omitting the factor Xa digest (not shown). solved, evidence for a specific platelet membrane protein functioning to accelerate migration of PL between membrane leaflets at increased cytosolic [Ca 2ϩ ] has been reported (12), similar to the proposed role of PL scramblase in red blood cells (10,11). It was thus of interest to determine whether mRNA for this protein is expressed in nucleated cells where PL scramblase-like activity has been observed. As shown by Fig. 5, Northern blotting with PL scramblase cDNA revealed transcripts of ϳ1.6 and ϳ2.6 kilobases in all tissues and cell lines tested. Some tissue-to-tissue and cell line variability in the relative abundance of these two transcripts is apparent, the significance of which remains to be determined. Also notable was markedly reduced expression in HL-60 and the lymphoma lines Raji and MOLT-4, whereas abundant message was detected in spleen, thymus, and peripheral leukocytes. In addition to the transformed cell lines shown, mRNA for PL scramblase was also confirmed in human umbilical vein endothelial cells (not shown). Whereas these data imply that the same protein identified as mediating accelerated transbilayer flipflop of the erythrocyte membrane PL also plays a similar role in the plasma membrane of platelets, leukocytes, and other cells, actual confirmation for this role of PL scramblase awaits analysis of a cell line that is selectively deficient in this protein. In Scott syndrome, a bleeding disorder related to an inherited deficiency of plasma membrane PL scramblase function, erythrocytes and other cells deficient in PL scramblase activity were found to contain normal amounts of the PL scramblase protein (11). 2 Furthermore, despite the apparent deficiency in Scott syndrome cells of endogenous PL scramblase function, when PL scramblase protein from these cells was purified and reconstituted in proteoliposomes containing exogenous PL, it exhib-ited normal Ca 2ϩ -dependent PL mobilizing activity (11). This suggests that in addition to the known regulation by intracellular [Ca 2ϩ ], the activity of PL scramblase in the plasma membrane is regulated by other as yet unidentified membrane or cytoplasmic component(s).