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Originally published In Press as doi:10.1074/jbc.M308210200 on August 21, 2003

J. Biol. Chem., Vol. 278, Issue 48, 47979-47986, November 28, 2003
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Processing of Surfactant Protein C Requires a Type II Transmembrane Topology Directed by Juxtamembrane Positively Charged Residues*

Surafel Mulugeta{ddagger} and Michael F. Beers

From the Lung Epithelial Cell Biology Laboratories, Pulmonary and Critical Care Division, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-4318

Received for publication, July 28, 2003 , and in revised form, August 14, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Surfactant protein C (SP-C) is a lung-specific protein that is synthesized as a 21-kDa integral membrane propeptide (pro-SP-C) and proteolytically processed to a 3.7-kDa secretory product. Previous studies have shown that palmitoylation of pro-SP-C is dependent on two N-terminal juxtamembrane positively charged residues. We hypothesized that these residues influence modification of pro-SP-C by directing transmembrane orientation. Double substitution mutation of these juxtaposed residues from positive to neutral charged species resulted in complete reversal of transmembrane orientation of pro-SP-C and total abrogation of post-translational processing. Mutation of a single residue resulted in mixed orientation. Protein trafficking studies in A549 cells showed that while the double mutant was retained in the endoplasmic reticulum, single mutants produced a mixed pattern of both endoplasmic reticulum (double mutant-like) and vesicular (wild type-like) expression. Our study demonstrates the crucial role juxtamembrane positively charged residues play in establishing membrane topology and their influence on the trafficking and processing of pro-SP-C. Moreover this study provides a likely precedent for a mechanism in disorders associated with mutations in the membrane-flanking region of integral membrane proteins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The alveolar form of surfactant protein C (SP-C)1 is a small hydrophobic peptide found in pulmonary surfactant, a biochemically heterogeneous complex of phospholipids and proteins that functions to prevent atelectasis by reducing surface tension at low lung volume. SP-C is synthesized exclusively in alveolar type II cells as a 21-kDa bitopic propeptide (pro-SP-C) with a type II orientation (Ncytosol/Clumen) and a 24° average angle of orientation with respect to the phospholipid bilayer (1) (see Fig. 1).



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  		FIG. 1.
Amino acid sequence of the human SP-C proprotein showing cytosolic, transmembrane, and luminal domains. The juxtamembrane lysine and arginine residues (lower inset), the palmitoylation site at Cys28 and Cys29 (upper inset), and the targeting motif (10MESPPDYSA18) to distal vesicles are shown. The position of the secreted mature SP-C relative to its precursor protein containing 197 amino acids is shown. The mature SP-C includes the entire transmembrane domain and a portion of the membrane-flanking cytosolic domain. Amino acid nomenclature is based on the published SP-C sequence (25).

 
SP-C represents a unique entity with respect to its biosynthesis and secretion. It undergoes extensive post-translational processing as it is trafficked through the biosynthetic pathway from the Golgi complex via small vesicles and multivesicular bodies to lamellar bodies where the mature form is stored prior to secretion to the alveolar lumen (2). As with other integral membrane proteins, its proper post-translational processing requires coordinated processes including targeting of the nascent protein to the endoplasmic reticulum (ER) membrane, insertion of the transmembrane domains in the correct orientation, proper conformation of tertiary and quaternary structures, and accurate trafficking of the protein to subcellular compartments. To date, SP-C is the only peptide known to be synthesized as an integral membrane protein and secreted via a regulated pathway as a luminal protein (2). This contrasts with other membrane-associated precursor proteins that remain limiting membrane-bound until their incorporation into the plasma membrane where they are proteolytically cleaved and released (2, 3).

The importance of proper conformation for protein processing and the deleterious effects of misfolding in SP-C and other proteins have been described (4–7). Diverse human disorders such as Alzheimer's disease, Huntington disease, cystic fibrosis, amyotrophic lateral sclerosis, {alpha}1-antitrypsin deficiency, and Parkinson disease have been shown to arise from protein misfolding and are now grouped together under the category of conformational diseases (4, 7). Recent reports have identified more than 12 mutations in the gene encoding SP-C in association with various interstitial lung diseases (8–10). Altered conformation as a result of one of these mutations has been shown to result in aggresome formation (ubiquitin/proteasome-bound perinuclear protein aggregates) and imparts a dominant negative effect on wild type protein processing (6).

In contrast to conformational factors, direct experimental evidence of the effects of topology on protein processing is lacking. The specific transmembrane orientation of the protein allows for cytosol- or lumen-specific modification(s) and target signal interactions. The topologic and targeting properties of a protein are primarily directed by specific signal domain(s) bordering hydrophobic transmembrane sequences of the protein (11–13). Studies suggest that N-terminal membrane-flanking positively charged residues play an essential role in influencing membrane topology (14–17).

Within the mature portion of pro-SP-C (Phe24–Leu58), a functional signal anchor domain mediates translocation of the nascent polypeptide to the ER, whereas the N-terminal domain contains a signal motif (10MESPPDYSA18) (see Fig. 1) that directs targeting to distal subcellular compartments (18, 19). In some models, the N-terminal and mature SP-C domains are sufficient for SP-C secretion (19). However, mutations producing structural or conformational alterations of the C-terminal domain of pro-SP-C result in abnormal trafficking of the peptide (6, 20).

The 35-amino acid sequence located N-terminal relative to the transmembrane domain of both rat and human pro-SP-C contains two adjacent post-translationally palmitoylated cysteine residues (Cys28 and Cys29) (Fig. 1, upper inset) and two contiguous juxtamembrane positively charged residues (Lys34 and Arg35) (Fig. 1, lower inset). Substitution of these residues with uncharged glutamine residues results in significantly reduced palmitoylation (21). In addition, fluorescence microscopy revealed that the mutant isoform appeared to be retained in the ER. The study suggested that the lysine and arginine residues influence transport of pro-SP-C to compartments distal to the ER. However, the possibility that the orientation of pro-SP-C is altered by the mutation of lysine and arginine was not examined.

We report that the juxtamembrane positively charged lysine and arginine play an essential role in the determination of pro-SP-C transmembrane topology. Alteration of these residues by substitution mutation with neutrally charged residues resulted in profound changes of transmembrane orientation, trafficking, and processing of the protein. Moreover mutations from positive to negative charges of these residues resulted in perinuclear aggregates of the proprotein with ubiquitin co-localization, a characteristic similar to aggresome formation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Epitope-specific Primary Antibodies—Antisera raised against specific domains of rat and human pro-SP-C were described previously (22). Polyclonal antiserum raised against the Met10–Glu23 domain of rat pro-SP-C recognizes N-terminal flanking peptides of both human and rat pro-SP-Cs. Polyclonal antibodies raised against the rat (Ser149–Ser166) and human (Gly162–Gly174) domains recognize the C-terminal proprotein of rat and human pro-SP-C, respectively.

SP-C cDNA Expression—Wild type pro-SP-C expression of human (1–197 amino acids) and rat (1–194 amino acids) in pEGFPC1 and pcDNA3 vectors has been described previously (6, 23). For creation of mutant proteins, single or double substitution mutation was achieved by overlap extension PCR with a two-round, four-primer technique as previously published (24) using wild type pro-SP-C cDNAs as templates. The first round involves the amplification of two overlapping PCR products, products A and B. Product A ranges from the 5' start site to the lysine- and arginine-encoding region, while product B comprises nucleotides from the lysine- and arginine-encoding region to the 3' untranslated poly(A) tail. The second round (splicing by overlap extension reaction) joined these two overlapping products together to produce cDNAs encoding mutant SP-C proproteins. A total of 20 primers were used. Six of the oligonucleotides (containing restriction sites for insertion into vectors) used for the 5' (product A) and 3' (product B) ends of human and rat pro-SP-C have been described previously (6, 23). Fourteen of the oligonucleotides that were used to generate mutant products correspond to nucleotides 115–144 of human and 100–129 of rat cDNAs of sequences published by Warr et al. (25) and Fisher et al. (26) (GenBankTM accession numbers NM_003018 [GenBank] and NM_017342 [GenBank] , respectively). Forward oligonucleotides that were used to generate type B products are as follows, and their complement sequences were used as reverse primers to make type A products: for human: pro-SP-CK34A,R35A, 5'-CCAGTGCACCTGGCTGCTCTTCTTATCGTG-3'; pro-SP-CK34A, 5'-CCAGTGCACCTGGCTCGCCTTCTTATCGTG-3'; pro-SP-CR35A, 5'-CCAGTGCACCTGAAAGCTCTTCTTATCGTG-3'; pro-SP-CK34E,R35E, 5'-CCAGTGCACCTGGAGGAGCTTCTTATCGTG-3'; for rat: pro-SP-CK34A,R35A, 5'-CCCGTGCATCTCGCTGCTCTTCTCATCGTG-3'; pro-SP-CK34A, 5'CCCGTGCATCTCGCTCGCCTTCTCATCGTG-3'; pro-SP-CR35A,5'-CCCGTGCATCTCAAAGCTCTTCTCATCGTG-3'.

Transient transfection of lung epithelial A549 cells has been described previously (24). Cells grown to 70% confluence on glass coverslips in 35-mm plastic dishes were transiently transfected with enhanced green fluorescence protein (EGFP)·pro-SP-C constructs (10 µg/dish) by CaPO4 precipitation. The medium was replaced at 24 h. Cells were maintained for up to 48 h after introduction of plasmid DNA.

Immunocytochemistry—Co-localization studies were performed by immunostaining plated cells that were fixed by immersion of coverslips in 4% paraformaldehyde. Following permeabilization, cells were immunolabeled with primary antibodies for 1 h at room temperature at the following dilutions: anti-calnexin (Stressgene, Victoria, Canada), 1:200; anti-CD63 (Immunotech, Marseilles, France), 1:100; anti-ubiquitin (Chemicon, Temecula, CA), 1:100. Texas Red-conjugated secondary goat anti-mouse IgG monoclonal or secondary goat anti-rabbit IgG polyclonal antibodies (Jackson Immunoresearch Laboratories, West Grove, PA) at 1:200 dilution were used for visualization. Fluorescence images of air-dried and Mowiol-mounted slides were viewed on an Olympus I-70 inverted fluorescent microscope. Fluorescence and phase images were captured using a Hamanatsu 12-bit coupled-charged device camera. Image processing and overlay analysis were performed using IMAGE 1 software (Universal Imaging, West Chester, PA).

Isolation of Integral Membrane Proteins: Carbonate Method—Integral membrane proteins were separated from peripherally associated membrane proteins as described previously (27) with modifications. Pellets collected by scraping cells from 35-mm dishes and centrifuged at 300 x g were resuspended with ice-cold phosphate-buffered saline (PBS) (137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM KH2PO4, pH 7.4) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1.5 µg/ml aprotinin) and sonicated on ice three times with 20-s bursts at 50 watts. The sonicate was centrifuged at 9,000 x g at 4 °C for 30 s to remove nuclei. The nuclei-free suspension was centrifuged at 4 °C for 1 h at 100,000 x g to separate cytosolic (supernatant) from integral/peripheral membrane (pellet) proteins. Following removal of the supernatant the pellet was resuspended with 100 mM sodium carbonate, pH 11.5, incubated at 0 °C for 30 min, and centrifuged at 4 °C for 1 h at 100,000 x g to separate the integral (pellet) from the peripherally associated membrane proteins. Samples were prepared for immunoblotting by resuspending pellets in lysis buffer (50 mM Tris, 190 mM NaCl, 6 mM EDTA, 2% Triton X-100, pH 7.4) containing protease inhibitors or by precipitating supernatants with 10% (w/v) trichloroacetic acid on ice, pelleting by centrifugation at 4 °C for 10 min at 12,000 x g, washing twice with acetone, drying with N2, and dissolving in lysis buffer.

Immunoblotting—Samples in lysis buffer were separated by 12% SDS-PAGE and transferred to nitrocellulose membrane. Immunoblotting was done using successive incubations with primary polyclonal GFP antiserum (Molecular Probes, Eugene, OR) (1:5000) for 1 h and goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:10,000) for 1 h at room temperature. Bands were visualized by enhanced chemiluminescence using a commercially available kit (Amersham Biosciences). Fluorescence images were produced either by exposure to film or by direct acquisition using the Kodak 440 Imaging System.

Membrane Orientation Analysis—In vitro transcription/translation of wild type and mutant pro-SP-C were done using the TNT coupled rabbit reticulocyte lysate system (Promega, Madison, WI). These reactions were performed in the presence of a [35S]cysteine/methionine translabel (Amersham Biosciences) according to the manufacturer's instructions at 30 °C for 1 h in the absence or presence of canine pancreatic microsomal membranes (Promega). Determination of membrane orientation of translated products in microsome vesicles was performed at least three times using each of two different techniques.

Protease Protection Assay—Protection of membrane protein by microsomes from proteinase K digestion was performed after in vitro translation of pro-SP-C (in pcDNA3 plasmid) in the presence of microsomes. Proteinase K was added to a final concentration of 0.1 mg/ml followed by incubation at 0 °C for 60 min as recommended by the manufacturer (Roche Applied Science). Phenylmethylsulfonyl fluoride was added to a final concentration of 4 mM to stop the digestion. For subsequent immunoprecipitation, sample volume was adjusted to 500 µl using ice-cold lysis buffer followed by the addition of 10 µl of primary antibody and incubation overnight at 4 °C with end-to-end rotation. 50 µl of magnetic protein A-microbeads (Multenyi Biotec, Auburn, CA) were added and incubated for 3 h at 4 °C with end-to-end rotation to magnetically label the immune complex. The mixtures were loaded onto microcolumns of the µMACS magnetic separator and washed four times with ice-cold wash buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, pH 7.5) containing 0.1% Triton X-100 and washed twice with ice-cold wash buffer. Finally samples were eluted with 50 µl of preheated 95 °C SDS loading buffer. Proteins were resolved by 12% SDS-PAGE and transferred to nitrocellulose membrane for autoradiography. Autoradiography was performed using both x-ray films and a PhosphorImager using Quantity One computer software (Bio-Rad).

Epitope-specific Pull-down Assay—Immunobinding of microsome-inserted proteins to evaluate orientation was performed after in vitro translation of pro-SP-C in the presence of microsomes. Following translation, sample volumes were adjusted to 500 µl with ice-cold PBS followed by the addition of 10 µl of primary antibody and incubation of the samples overnight at 4 °C with end-to-end rotation. To separate microsome vesicles from the lysate (containing non-inserted protein· antibody complexes and unbound antibodies), samples were loaded on a cushion of 1 ml of dibutylphthalate (Sigma) on top of 1.5 ml of 0.50 M sucrose and centrifuged for 1 h at 100,000 x g using a swinging bucket rotor. Isolated microsomes recovered from the pellet were resuspended in 500 µl of lysis buffer, and antibody complexes were precipitated by incubation with 50 µl of magnetic protein A-microbeads for 3 h at 4 °C with end-to-end rotation. The mixtures were loaded into microcolumns on the µMACS magnetic separator, washed, prepared for electrophoresis, and autoradiographically processed as described above. Control analyses consisted of in vitro translation of pro-SP-C in the absence of microsomes utilizing lysis buffer instead of PBS during the primary antibody binding procedure and omitting the centrifugation step.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
To define the mechanism underlying the influence of juxtamembrane residues on SP-C biosynthesis, human and rat pro-SP-C cDNA clones were systematically altered by site-directed mutagenesis with juxtamembrane positively charged lysine (Lys34) and/or arginine (Arg35) residues substituted with either neutrally charged alanine (Ala) or negatively charged glutamate (Glu) residues to yield pro-SP-CK34A,R35A, pro-SP-CK34A, pro-SP-CR35A, or pro-SP-CK34E,R35E mutant constructs. The clones were inserted into either a pcDNA3 vector to evaluate topological orientation using an in vitro system or fused with an EGFP expression vector to characterize biosynthesis (trafficking, subcellular compartmentalization, and processing) in an alveolar epithelial cell line (A549 cells). Experiments were conducted in parallel with both human and rat mutant isoforms unless otherwise indicated. All data obtained with the human isoforms were identical to those of the rat; therefore, only data using human SP-C are shown.

Differential Trafficking of Wild Type and Mutant Proteins— Transient transfection of EGFP plasmid alone (control) in A549 cells showed a diffuse fluorescence distribution throughout the cell indicative of non-targeted protein expression (Fig. 2a). Wild type EGFP·SP-C was sorted to punctate ring-like cytoplasmic vesicles (Fig. 2b), which has been shown previously to be a characteristic of successful membrane protein trafficking to late secretory compartments (6). Wild type EGFP·SP-C also co-localized with CD63 (data not shown), a marker antigen associated with lamellar bodies and multivesicular bodies of alveolar type II cells. In contrast, the double mutant form where neutral amino acids were substituted for basic residues (pro-SP-CK34A,R35A) displayed a reticular expression pattern (Fig. 2c) consistent with ER localization. Calnexin, an ER-specific marker, strongly co-localized with the EGFP-tagged mutant isoform confirming that pro-SP-CK34A,R35A was retained in the ER (Fig. 3A).



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  		FIG. 2.
EGFP wild type and mutant pro-SP-C chimeric proteins are trafficked differentially. Fluorescent microscopy of A549 cells transfected with EGFP vector alone (a), EGFP·pro-SP-CWT (b), and EGFP·pro-SP-CK34A,R35A (c) is shown. Three distinct trafficking patterns are apparent: diffuse cytosolic appearance of vector alone (a), ring-like display (inset) of vesicles of the wild type (b), and reticular pattern of the mutant isoforms (c). Plasmid and/or constructs used for each transfection are shown above each panel. Bar, 5 µm.

 



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  		FIG. 3.
EGFP·pro-SP-CK34A,R35A mutant proteins are retained in the ER and are not post-translationally processed. A, fluorescence images of A549 cells transfected with EGFP·pro-SP-CK34A,R35A (left), immunostained with calnexin (middle), and merged picture (right) showing co-localization of the mutant form of SP-C and calnexin. B, anti-GFP immunoblots of membrane fractions from A549 cells transfected with EGFP alone (lanes 1–4), EGFP·pro-SP-CWT (lanes 5–8), and EGFP·pro-SP-CK34A,R35A (lanes 9–12). Blots of whole cell lysate (lanes 1, 5, and 9), postcentrifuge supernatant before (lanes 2, 6, and 10) and after (lanes 3, 7, and 11) sodium carbonate treatment of initial pellet, and postcentrifuge pellet (lanes 4, 8, and 12) products are shown. The primary product of EGFP protein alone migrates to 27 kDa, while primary products of pro-SP-CWT and mutant EGFP fusion proteins migrate to 48 kDa. Processed (lower doublet) and palmitoylated (upper band of top doublet) pro-SP-CWT products (lanes 5 and 8) are apparent. Plasmid and/or constructs used for each transfection are shown at the top of each panel.

 
When only one of the two positively charged juxtamembrane residues was mutated to alanine, a mixed pattern of expression was observed. As shown in Fig. 4A, expression of EGFP·pro-SP-CK34A resulted in the retention of some mutant protein in the calnexin-positive ER compartment (left panel) with another fraction accumulating in CD63-positive cytoplasmic vesicles (right panel) comparable to the wild type isoform (6). Similar results were obtained using an EGFP·pro-SP-CR35A plasmid (data not shown). In contrast to the above findings, when both residues were substituted to negatively charged glutamate (EGFP·pro-SP-CK34E,R35E), expression resulted in formation of perinuclear-restricted, ubiquitin-positive inclusion bodies (Fig. 4B). This result is consistent with recent reports of the characteristics of proteins directed to ubiquitin/proteasome degradation pathways and aggresome formation (6, 20).



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  		FIG. 4.
EGFP·pro-SP-CK34A is partially trafficked to post-Golgi vesicles, while EGFP·pro-SP-CK34E,R35E is directed to perinuclear ubiquitinated inclusion bodies. A, A549 cells transfected with the EGFP fusion construct with a single substitution mutation (pro-SP-CK34A) (top row) were immunostained with calnexin (left middle) and CD63 (right middle). Both markers appear to co-localize with the mutant (merged images), which indicates that this mutant is targeted to both ER and vesicular compartments. B, ubiquitin immunostaining of A549 cells transfected with an EGFP construct that contained negatively charged residues (pro-SP-CK34E,R35E) in place of the positively charged residues is shown. Transfected mutants show perinuclear inclusion bodies with ubiquitin co-localization. Only human pro-SP-C constructs were used for this experiment. All figures are representative of at least three separate experiments. Constructs used for each transfection are shown at the top of each panel. N, nucleus. Bar, 5 µm.

 
Proprotein Mutation Results in the Abrogation of Post-translational Processing—Immunoblotting of the membrane fractions of transfected cells with anti-GFP revealed processing of wild type SP-C as shown by bands (Fig. 3B, lanes 5 and 8) corresponding to the predicted Mr of the primary translation product of the fusion protein (48,000), palmitoylated product (upper band of top doublet), and smaller intermediate cleaved products as previously reported (28, 29). In contrast, EGFP·pro-SP-CK34A,R35A was not processed to either cleaved intermediates or palmitoylated product as demonstrated by the single band at Mr of 48,000 (Fig. 3B, lanes 9 and 12).

In membrane association assays, no translation product was detected in a post-100,000 x g centrifuged cytosolic fraction (Fig. 3B, lane 10). In addition, Na2CO3 treatment of the membrane fraction did not release any peripherally associated products (Fig. 3B, lane 11) indicating that EGFP·pro-SP-CK34A,R35A remains an integral membrane protein and that lysine and arginine residues do not function as stop transfer signals that participate in the anchoring of pro-SP-C to the ER membrane.

Effect of Juxtamembrane Amino Acid Residues on Transmembrane Protein Topology—The role of juxtamembrane amino acid residues in determining transmembrane topology was examined using an in vitro protease protection assay. After in vitro transcription/translation of wild type or mutant [35S]cysteine/methionine-translabeled SP-C proproteins in the presence or absence of microsomes (to allow for insertion of the proprotein into microsomal membranes), samples were tested for resistance to proteolytic cleavage with proteinase K. Immunoprecipitation of samples with an antibody recognizing the pro-SP-C C terminus (C-term) revealed that a portion of wild type pro-SP-C (pro-SP-CWT) was protected from proteolysis inside the microsome vesicle producing a band at the predicted size of 17 kDa (Fig. 5, lane 5, arrow). This result indicates that proper membrane insertion of pro-SP-CWT is not altered by the in vitro transcription/translation method. In contrast, the 17-kDa portion of pro-SP-CK34A,R35A was not protected by the microsomes as observed by the absence of an identifiable band in the N-terminal (NPro) (Fig. 5, lane 9) or C-term (Fig. 5, lane 11) antibody-precipitated products. If topology for the mutant protein had been reversed, the predicted protected size would be 6.2 kDa recognized by NPro. This was below the resolvable power of electrophoresis due to the relatively large size (Mr 7,000 or below) (Fig. 5, lanes 3, 5, 7, 9, and 11) of cleaved fragments of pro-SP-C by proteinase K. Repetition of these experiments using trypsin produced identical results (data not shown).



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  		FIG. 5.
Wild type and mutant pro-SP-Cs show topological differences. Gel electrophoresis autoradiography of in vitro expressed and immunoprecipitated products of [35S]cysteine/methionine-translabeled pcDNA3 pro-SP-C constructs. The primary translation products at 21 kDa are shown. *, multimeric pro-SP-C products above the primary translation product are artifacts of the in vitro translation system used. pcDNA3 vector only (lane 1), pro-SP-CWT (lanes 2–5), and pro-SP-CK34A,R35A (lanes 6–11) constructs in the presence (lanes 4, 5, 8, 9, and 11) and absence (lanes 1, 2, 3, 6, 7, and 10) of canine pancreatic microsomes (CPM) were treated with proteinase K (lanes 3, 5, 7, 9, and 11). Lysates were immunoprecipitated with C-term (lanes 1–5, 10, and 11) and NPro (lanes 6–9) SP-C antibodies. Microsome-protected product of 17 kDa (arrow) after proteinase K treatment is observed only in the wild type isoform (lane 5); bottom bands ~7 kDa and below are the electrophoresis migration front representing enzymatically cut fragments of pro-SP-C.

 
To corroborate this apparent change of orientation, an alternative approach to evaluate transmembrane orientation was carried out using wild type or mutant pro-SP-Cs translated in vitro in the presence of microsome vesicles followed by incubation with epitope-specific antibodies. The antibodies were first allowed to bind to inserted proteins on intact microsomes. The pro-SP-C·antibody·microsome complexes were separated from unbound antibodies and uninserted proteins. Subsequently the complexes were precipitated with magnetic protein A-agarose beads (see "Experimental Procedures"). Under these conditions the NPro (Fig. 6, lane 3) but not the C-term antibody (Fig. 6, lane 9) associated with pro-SP-CWT, consistent with a type II orientation. In contrast, pro-SP-CK34A,R35A achieved a type III orientation (Ccytosol/Nlumen) and associated with the C-term (Fig. 6, lane 9) but not the NPro antibody (Fig. 6, lane 3). SP-C proproteins with single mutations, pro-SP-CK34A and pro-SP-CR35A, were immunoprecipitated with both N- and C-terminal antibodies (Fig. 6, lanes 5 and 7) indicating a mixed orientation.



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  		FIG. 6.
Orientation of mutant proproteins is inverted. Gel electrophoresis autoradiography of in vitro expressed [35S]cysteine/methionine-translabeled pcDNA3 pro-SP-C constructs. Integral membrane proteins were immunobound and separated using an epitope-specific pull-down assay. Vector only (lane 1), pro-SP-CWT (lanes 2 and 3), pro-SP-CK34A (lanes 4 and 5), pro-SP-CR35A (lanes 6 and 7), and pro-SP-CK34A,R35A (lanes 8 and 9) in the presence (lanes 3, 5, 7, and 9) and absence (lanes 1, 2, 4, 6, and 8) of microsomes. Samples were immunobound with NPro (upper panel) and C-term (lower panel) SP-C antibodies. *, multimeric pro-SP-C products above the primary translation product are artifacts of the in vitro translation system used. CPM, canine pancreatic microsomes.

 
Taken together, these in vitro experiments clearly demonstrate that the juxtamembrane lysine and arginine residues affect membrane orientation of pro-SP-C. While single mutation of either one of the residues produced mixed orientation, double mutation resulted in complete reversal of orientation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The effect of a mutation on membrane topology and its influence on protein processing are often overlooked as a potential mechanism for abnormal protein biosynthesis since it is generally assumed that alteration of transmembrane protein topology requires extensive sequence changes (30). Here we show that a single amino acid substitution within a membrane-flanking domain can profoundly alter transmembrane protein topology. Our study further suggests that proper trafficking and processing of SP-C requires cytosolic localization of the targeting motif (10MESPPDYSA18) of the precursor protein. Moreover the close association between processing and trafficking provides a paradigm for predicting abnormal trafficking of a protein by analyzing modification and processing patterns of the protein using gel electrophoresis data.

The membrane orientation of pro-SP-C was determined by two independent methods. The first is a "protease protection" assay that has been widely used for membrane protein topology determination. As shown in Fig. 5, we identified a proteinase K-digested fragment of Mr 17,000 in wild type SP-C consistent with protection of C-terminal fragment in the microsome lumen. However, due to the size of protease-digested peptide fragments and technical limitation of SDS-PAGE, this assay is limited to the resolution of peptides with molecular mass of more than ~7 kDa thus impeding the ability to document a protected fragment for the double mutant. Using a second, independent method, "epitope-specific pull-down" assays confirm the reversal of orientation in the double mutant. This technique represents a novel method that identifies and precipitates only membrane-inserted proteins containing epitopes recognized by specific antibodies. In addition, the use of a dibutylphthalate cushion during centrifugation prevents mixing of aqueous layers thus limiting contamination with unbound peptides and antibodies. This method not only leaves the native protein intact but also eliminates protease fragment interference.

Using these methods, the data clearly show double substitution mutation of lysine and arginine residues of pro-SP-C to alanine results in the inversion of protein orientation thereby restricting the targeting motif to the lumen of the ER (Fig. 7). Several studies using both prokaryotic and eukaryotic proteins have demonstrated that N-terminal membrane-flanking positively charged residues function as signals for establishing topological orientation (14–16, 31–33). However, none of these experiments have shown an absolute inversion of total protein population suggesting additional factors other than charged residues may be required to establish uniform orientation. Furthermore a number of topology hypotheses have been proposed based on analyses of known membrane protein sequences (32, 34, 35). One of these hypotheses, the "charge difference" hypothesis (35), states that charges within 10–15 residues of each side of the membrane-flanking domain influence orientation and that the more positive charge is cytoplasmic. Our study, using mammalian proteins, shows that substitution of two juxtamembrane positively charged residues to neutral charges inverted the orientation of the total SP-C pro-protein population. We also show mixed orientation of the protein with single substitution mutation. Our data thus suggest that charges of the membrane-flanking domains are the primary determinants of pro-SP-C topology. As shown in Table I, SP-C proprotein membrane orientation appears to adhere to the charge difference hypothesis when the 10 residues on each side of the membrane-flanking domain are taken into account.



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  		FIG. 7.
Double mutation of juxtamembrane positively charged residues to neutral charges results in complete inversion of pro-SP-C orientation. Diagrammatic representation of pro-SP-CWT and pro-SP-CK34A,R35A topology demonstrating that double mutation of lysine and arginine to alanine reverses the orientation completely from type II to type III is shown.

 


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  		TABLE I
Membrane integration, topology, and trafficking of pro-SP-C are influenced by the charges of membrane-flanking domains

 
The membrane association assay in A549 cells (Fig. 3B) demonstrates that the two juxtamembrane positively charged residues do not function as stop transfer signal and that pro-SP-C mutants remain membrane-bound. Moreover partially processed EGFP·pro-SP-CWT, identified by N-terminal EGFP domain, is also membrane-bound, and no cytosolic EGFP is detected indicating the absence of N-terminal cleavage of the propeptide. This finding differs from those of in vivo experiments and studies in freshly isolated alveolar type II cells that showed that processing of pro-SP-C entails four proteolytic cleavages in compartments distal to Golgi (2). While the C-terminal flanking propeptide is initially cleaved in succession in early compartments, the two N-terminal cleavages take place in distal sites including multivesicular bodies and lamellar bodies. Based upon these observed differences in pro-SP-C processing between alveolar type II cells and A549 cells, it is apparent that A549 cells, unlike alveolar type II cells, do not process the propeptide to its mature form. As shown in Fig. 3B and also as described previously (6, 20, 23), wild type pro-SP-C is partially processed by two-step cleavage of the C terminus in A549 cells (Fig. 3B, lanes 5 and 8, lower doublets). The presence of ring-like vesicles in transfected cells (Fig. 2B, inset) and the absence of released EGFP at Mr 27,000 (Fig. 3B) support previous findings that the final steps of processing are absent in this cell type leaving the partially processed propeptide bound to the membrane with an intact N-terminal domain.

In addition to being an integral bitopic membrane protein, pro-SP-C is a true lipopeptide, containing palmitic acid held in thioester linkage at Cys28 and Cys29. Biosynthesis of palmitoylated SP-C precursors has been demonstrated in murine fetal lung and Chinese hamster ovary cells transfected with a human SP-C cDNA and is an early post-translational modification that occurs in the Golgi (36, 37). Recently ten Brinke et al. (21) demonstrated that the palmitoylation of pro-SP-C could be blocked in vitro by mutagenesis of Lys34 and Arg35. Similar to our study, the mutants were retained in the ER. The defect in processing observed in these two studies is unlikely to be related to palmitoylation. Transfection of heterologous fusion proteins consisting of EGFP attached to the N terminus of either wild type rat pro-SP-C (EGFP·rSP-CWT) or mutant forms containing point mutations of both residues Cys28-Cys29 to either glycine (EGFP·rSP-CC28G,C29G) or serine (EGFP·rSP-CC28S,C29S) were trafficked to cytoplasmic sites in a fashion identical to EFGP·rSP-CWT suggesting that these moieties are not required for normal biosynthesis (37). Taken together, the defect in SP-C trafficking observed with juxtamembrane charge mutations is not due to secondary effects of palmitoylation but likely to be a primary defect in trafficking pro-SP-C to the Golgi imparted by an altered transmembrane topology.

The double mutant isoform containing negatively charged glutamate residues (Fig. 4B) showed a distinct trafficking behavior from either the double or single mutant isoforms of neutrally charged residues (Figs. 3A and 4A). This observation indicates that abnormal trafficking of the double glutamate mutant pro-SP-C results from a different process from that which determines topology. The juxtanuclear localization of ubiquitinated inclusion bodies of this mutant isoform suggests a common pathway for misfolded membrane proteins comprised of the ubiquitin/proteasome degradation pathway and aggresome formation (perinuclear localized bodies that are believed to be formed due to an overload of misfolded proteins) (38, 39). Such aggresome formations are observed with mutants of pro-SP-C including the {Delta}exon 4 mutation associated with interstitial lung disease (6, 20). In our study, the elevated charge difference (Table I) between cytosolic and luminal membrane-flanking domains in mutants containing negatively charged juxtamembrane residues may have resulted in the failure of correct translocation and insertion of the glutamate mutant proprotein into the lipid bilayer. This would lead to the exposure of hydrophobic side chains to the aqueous cytosolic environment thereby compelling the proprotein to adopt an alternative, misfolded conformation. Interaction between misfolded proteins may lead to the development of aggregates and subsequent aggresome formation.

Inappropriate processing due to mutations in certain transmembrane proteins is known to be associated with pathologic abnormalities. Some of these proteins, similar to SP-C, are synthesized as proproteins and undergo processing to achieve their mature and biologically active forms as they are trafficked through different subcellular compartments. Proteins such as the amyloid precursor protein, angiotensin-converting enzyme, and peripheral myelin protein 22 remain membrane-bound until released as their mature forms by post-translational proteolytic cleavage at the plasma membrane (3). The amyloidogenic fragments in Alzheimer's disease (40), the peripheral myelin protein 22 mutation in Charcot-Marie-Tooth disease type 1A (41, 42) and the insertion/deletion polymorphism of the angiotensin-converting enzyme gene in various cardiovascular diseases (43) (including coronary atherosclerosis (44), ischemic stroke (45), and malignant hypertension (46)) are all believed to be consequences of mutations leading to abnormal processing of their respective precursor proteins.

Similarly a number of mutations in the SP-C gene have been identified in association with a variety of interstitial lung diseases (9, 10). These heterozygous gene mutations not only include various single amino acid substitutions or deletions in the C-terminal domain distal to the transmembrane anchor but also a number of single amino acid substitutions in both the N- and C-terminal membrane-flanking regions. One of these mutations, a Pro30 to leucine mutation has a characteristic of an ER-retained and a predominantly non-palmitoylated protein (28). Given the charge to orientation sensitivity of pro-SP-C demonstrated in this study, these mutations in the membrane-flanking regions are likely to affect pro-SP-C topology and contribute to the abnormal processing of SP-C.

Data presented in this study together with the experimental findings in both prokaryotes and eukaryotes (14, 15, 32, 47) suggest a conserved mechanism for establishing transmembrane protein topology. Although our study shows a complete reversal of orientation, most mutations may result in subtle topological changes. For example, instead of directing orientation, some amino acids may determine the relative position of the peptide in the lipid bilayer (17, 48) and hence may affect modification processes. In addition, since post-translational modifications such as glycosylation contribute to membrane protein topology (47), palmitoylation may have a similar role. Taken together, these observations suggest that when investigating mechanisms for the growing number of interstitial lung diseases associated with SP-C mutations or other pathological disorders associated with protein mutations in the membrane-flanking region, topological alteration should be considered as one potentially significant contributing factor.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants HL-19737, P50-HL56401, and HL74064 (to M. F. B.) and American Lung Association Dalsemer Research Grant DA-188-N (to S. M.). 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. Back

{ddagger} To whom correspondence should be addressed: Pulmonary and Critical Care Division, University of Pennsylvania, Abramson Research Center, Room 1015 Hb, 3615 Civic Center Blvd., Philadelphia, PA 19104-4318. Tel.: 215-746-2920; Fax: 215-573-4469; E-mail: mulugeta{at}mail.med.upenn.edu.

1 The abbreviations used are: SP-C, surfactant protein C; pro-SP-C, SP-C precursor protein; rSP-C, rat SP-C; ER, endoplasmic reticulum; GFP, green fluorescence protein; EGFP, enhanced GFP; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We thank M. Koval for critical reading and invaluable suggestions, S. J. Russo for assistance with the production of cDNA constructs, and C. D. Campbell for editorial expertise.



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 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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