Secretion of Surfactant Protein C, an Integral Membrane Protein, Requires the N-terminal Propeptide

Abstract

Proteolytic processing of surfactant protein C (SP-C) proprotein in multivesicular bodies of alveolar type II cells results in a 35-residue mature peptide, consisting of a transmembrane domain and a 10-residue extramembrane domain. SP-C mature peptide is stored in lamellar bodies (a lysosomal-like organelle) and secreted with surfactant phopholipids into the alveolar space. This study was designed to identify the peptide domain of SP-C required for sorting and secretion of this integral membrane peptide. Deletion analyses in transiently transfected PC12 cells and isolated mouse type II cells suggested the extramembrane domain of mature SP-C was cytosolic and sufficient for sorting to the regulated secretory pathway. Intratracheal injection of adenovirus encoding SP-C mature peptide resulted in secretion into the alveolar space of wild type mice but not SP-C (−/−) mice. SP-C secretion in null mice was restored by the addition of the N-terminal propeptide. The cytosolic domain, consisting of the N- terminal propeptide and extramembrane domain of mature SP-C peptide, supported secretion of the transmembrane domain of platelet-derived growth factor receptor. Collectively, these studies indicate that the N-terminal propeptide of SP-C is required for intracellular sorting and secretion of SP-C. Proteolytic processing of surfactant protein C (SP-C) proprotein in multivesicular bodies of alveolar type II cells results in a 35-residue mature peptide, consisting of a transmembrane domain and a 10-residue extramembrane domain. SP-C mature peptide is stored in lamellar bodies (a lysosomal-like organelle) and secreted with surfactant phopholipids into the alveolar space. This study was designed to identify the peptide domain of SP-C required for sorting and secretion of this integral membrane peptide. Deletion analyses in transiently transfected PC12 cells and isolated mouse type II cells suggested the extramembrane domain of mature SP-C was cytosolic and sufficient for sorting to the regulated secretory pathway. Intratracheal injection of adenovirus encoding SP-C mature peptide resulted in secretion into the alveolar space of wild type mice but not SP-C (−/−) mice. SP-C secretion in null mice was restored by the addition of the N-terminal propeptide. The cytosolic domain, consisting of the N- terminal propeptide and extramembrane domain of mature SP-C peptide, supported secretion of the transmembrane domain of platelet-derived growth factor receptor. Collectively, these studies indicate that the N-terminal propeptide of SP-C is required for intracellular sorting and secretion of SP-C. surfactant protein B surfactant protein C platelet-derived growth factor receptor phosphate-buffered saline green fluorescent protein hemagglutinin Type II epithelial cells synthesize and secrete pulmonary surfactant, a complex mixture of phospholipids and proteins that reduces surface tension along the alveolar air-liquid interface at end respiration. The peptide components of surfactant, in particular surfactant protein B (SP-B1) and SP-C, are critical for surfactant film formation and function. Newborn infants and mice lacking SP-B have dramatically reduced pulmonary compliance and develop lethal, respiratory distress syndrome shortly after birth (1Whitsett J.A. Nogee L.M. Weaver T.E. Horowitz A.D. Physiol. Rev. 1995; 75: 749-757Crossref PubMed Scopus (161) Google Scholar, 2Nogee L.M. BBA Mol. Basis Dis. 1998; 1408: 323-333Crossref PubMed Scopus (51) Google Scholar). Disruption of the SP-B locus results in incomplete processing of pro-SP-C to its mature peptide, leading to deficiency of both SP-C and SP-B. SP-C null mice have normal levels of SP-B and survive with subtle changes in lung function but normal lung structure and surfactant pool sizes. 2S. Glasser, submitted for publication.2S. Glasser, submitted for publication. The importance of SP-C for normal lung function is inferred from experiments in which intratracheal administration of surfactant containing SP-C as the sole protein component to preterm animals restored lung function to values comparable with animals treated with native surfactant (3Hafner D. Beume R. Kilian U. Krasznai G. Lachmann B. Br. J. Pharmacol. 1995; 115: 451-458Crossref PubMed Scopus (55) Google Scholar, 4Hawgood S. Ogawa A. Yukitake K. Schlueter M. Brown C. White T. Buckley D. Lesikar D. Benson B. Am. J. Respir. Crit. Care Med. 1996; 154: 484-490Crossref PubMed Scopus (57) Google Scholar, 5Davis A.J. Jobe A.H. Häfner D. Ikegami M. Am. J. Respir. Crit. Care Med. 1998; 157: 553-559Crossref PubMed Scopus (102) Google Scholar). Collectively, these results suggest that SP-C and SP-B are functionally interchangeable with respect to biophysical activity. SP-C is synthesized by the alveolar type II epithelial cell as a 197-amino acid proprotein in which the mature peptide (residues 24–58) is flanked by N-terminal (residues 1–23) and C-terminal (residues 59–197) peptide domains. Unlike SP-B, SP-C is an integral membrane protein, which contains a single membrane-spanning domain located within the mature peptide (6Johansson J. Szyperski T. Curstedt T. Wuthrich K. Biochemistry. 1994; 33: 6015-6023Crossref PubMed Scopus (176) Google Scholar). The topology of the SP-C proprotein in the membrane is not clear, with reports of both type II (7Keller A. Eistetter H.R. Voss T. Schäfer K.P. Biochem. J. 1991; 277: 493-499Crossref PubMed Scopus (51) Google Scholar) and type III (8Vorbroker D.K. Voorhout W.F. Weaver T.E. Whitsett J.A. Am. J. Physiol. 1995; 13: L727-L733Google Scholar) orientations. SP-C proprotein is detected in endoplasmic reticulum, Golgi, and multivesicular bodies but not in lamellar bodies, the intracellular storage compartment for pulmonary surfactant (8Vorbroker D.K. Voorhout W.F. Weaver T.E. Whitsett J.A. Am. J. Physiol. 1995; 13: L727-L733Google Scholar, 9Voorhout W.F. Weaver T.E. Haagsman H.P. Geuze H.J. van Golde L.M.J. Microsc. Res. Technique. 1993; 26: 366-373Crossref PubMed Scopus (67) Google Scholar). Processing of the proprotein results in cleavage of the propeptides and generation of the 35-amino acid mature peptide that is detected only in the multivesicular body and lamellar body (8Vorbroker D.K. Voorhout W.F. Weaver T.E. Whitsett J.A. Am. J. Physiol. 1995; 13: L727-L733Google Scholar, 10Beers M.F. Lomax C. Am. J. Physiol. 1995; 13: L744-L753Google Scholar). Colocalization of both proprotein and mature peptide in the multivesicular body strongly suggests that processing of the SP-C precursor to the biologically active peptide occurs within this compartment. In order to promote absorption and spreading of surfactant lipids at the alveolar air-liquid interface, mature SP-C peptide must be secreted by the type II cell. The mature peptide consists of an extremely hydrophobic transmembrane domain and a 10–12-amino acid extramembrane domain that contains palmitoylated cysteines at positions 5 and 6 (residues 28 and 29 of the proprotein) in most species (11Curstedt T. Johansson J. Persson P. Eklund A. Robertson B. Löwenadler B. Jornvall H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2985-2989Crossref PubMed Scopus (181) Google Scholar, 12Johansson J. Persson P. Löwenadler B. Robertson B. Jornvall H. Curstedt T. FEBS Lett. 1991; 281: 119-122Crossref PubMed Scopus (44) Google Scholar, 13Stults J.T. Griffin P.R. Lesikar D.D. Naidu A. Moffat B. Benson B.J. Am. J. Physiol. 1991; 261: L118-L125PubMed Google Scholar). The mechanism underlying secretion of this integral membrane peptide is not clear but probably involves two discrete steps. The proprotein is first sorted to the multivesicular body that ultimately fuses with the lamellar body, a lysosome-related organelle. Sorting of integral membrane proteins to lysosomes and secretory granules is dependent upon information encoded in the cytosolic domain of the protein (14Hunziker W. Geuze H.J. Bioessays. 1996; 18: 379-389Crossref PubMed Scopus (237) Google Scholar, 15Marks M.S. Ohno H. Kirchhausen T. Bonifacino S.J. Trends Cell Biol. 1997; 7: 124-128Abstract Full Text PDF PubMed Scopus (277) Google Scholar), suggesting that either the N-terminal or C-terminal peptide domain of the proprotein plays an important role in sorting SP-C to lamellar bodies. Since SP-C mature peptide is detected only in the lumen of the lamellar body, a second step is required to relocate SP-C from the limiting membrane to the lumen of the multivesicular/lamellar body. It is likely that the N-terminal or C-terminal peptide domain also facilitates luminal internalization of SP-C. This study was designed to identify the compartment in which SP-C internalization occurs and the peptide domains required for sorting and secretion of SP-C into the airspace. Full-length human SP-C cDNA was cloned into pcDNA3 (Invitrogen, San Diego, CA). To generate SP-C deletion constructs (Fig. 2 A), SP-C fragments were amplified by polymerase chain reaction using specific primers to human SP-C containing 5′ SacI and 3′ SacII cleavage sites. Polymerase chain reaction fragments were subcloned into rEGFP vector (CLONTECH, San Jose, CA) and subjected to bidirectional sequencing to verify the fidelity of the polymerase chain reaction product throughout the SP-C coding sequence and the green fluorescent protein (GFP)/SP-C junction. PC12 cells (a gift from D. Cutler, University College London) were cultured as described previously (16Lin S. Akinbi H.T. Breslin J.S. Weaver T.E. J. Biol. Chem. 1996; 271: 19689-19695Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Cells were grown in T25 flasks until 80% confluent and transiently transfected with 6 μg of plasmid DNA and 60 μl of Fugene 6 (Roche Molecular Biochemicals) according to the manufacturer's instructions. After 48 h of culture, PC12 cells were labeled with 0.5 mCi of [35S]methionine/cysteine (Amersham Pharmacia Biotech) for 4 h. Cell lysates and media were immunoprecipitated exactly as described previously by Lin et al. (17Lin S. Weaver T.E. BBA Mol. Cell Res. 1996; PubMed Scopus Google Scholar) with 5 μl of an the SP-C N-terminal propeptide D.K. Nogee L.M. Whitsett J.A. Am. J. Physiol. 1995; PubMed Google Scholar). and were as previously described (17Lin S. Weaver T.E. BBA Mol. Cell Res. 1996; PubMed Scopus Google Scholar). PC12 cells were After h of culture, PC12 cells were transiently transfected with 2 μg of plasmid DNA and μl of Fugene 6 (Roche Molecular Biochemicals) according to the manufacturer's instructions. 48 h PC12 cells were with in with and with the SP-C N-terminal propeptide D.K. Nogee L.M. Whitsett J.A. Am. J. Physiol. 1995; PubMed Google Scholar) and CA) for h at Cells were and with and for h at After to 4 cells were for at 4 with the SP-C N-terminal D.K. Nogee L.M. Whitsett J.A. Am. J. Physiol. 1995; PubMed Google Scholar) or C-terminal peptide domain W.F. Weaver T.E. Haagsman H.P. Geuze H.J. van Golde L.M.J. Microsc. Res. Technique. 1993; 26: 366-373Crossref PubMed Scopus (67) Google Scholar), and to membrane Cells were with and with in at 4 After cells were and with and for h at cells were with and with and with was with a and using Type II cells were from mice as described by al. M. Am. J. Respir. Cell Mol. Biol. 1996; PubMed Scopus Google Scholar) with the cell was by of of into the lung by 0.5 of to cell were isolated as previously described M. Am. J. Respir. Cell Mol. Biol. 1996; PubMed Scopus Google Scholar) and to with μg of and μg of San Diego, CA) in 6 of by for h at were to type II cells and at for at 4 Type II cells were in media and at a of and to to for 4 h. Cells were with and transfected with a at with was and cells were cultured for h by with mature SP-C as described The sequence encoding or was amplified from cDNA by chain reaction of type II cell isolated from mice using specific primers that a sequence in the 5′ and a hemagglutinin in the 3′ The was by using primers to the SP-C cytosolic domain (residues of the the transmembrane domain (residues and an polymerase chain reaction fragments were cloned into the vector K. 1998; PubMed Scopus Google Scholar) and to the of the and were as described previously K. 1998; PubMed Scopus Google Scholar). were into wild type and SP-C (−/−) mice Glasser, were and with 2 of in containing G. J. C. J. J. M. P. Am. J. Respir. Cell Mol. Biol. PubMed Scopus Google Scholar) in a of or after lung was with and and the was for with CA) as described previously W.F. Weaver T.E. Haagsman H.P. Geuze H.J. van Golde L.M.J. Microsc. Res. Technique. 1993; 26: 366-373Crossref PubMed Scopus (67) Google Scholar). mice were with saline and surfactant was isolated by at for at 4 from mice was and and were isolated a as described previously M. T. W. Whitsett J.A. G. Jobe A.H. Am. J. Physiol. 1996; Scholar). bodies were isolated from of mice as previously described by et al. K. Am. J. Respir. Cell Mol. Biol. 1998; PubMed Scopus Google Scholar). of protein, by acid protein B.J. Biochem. PubMed Scopus Google Scholar), from and were by and with an the This study was designed to identify the peptide that sorting and secretion of SP-C. isolated type II cells are to sorting studies were in transiently transfected PC12 PC12 cells were transfected with wild type and labeled with and cell lysates and media were SP-C proprotein was detected in cell lysates but was not to the active peptide or secreted into the media To wild type was sorted to the regulated secretory PC12 cells were transfected with and with the SP-C proprotein and a for type proprotein with with sorting to the regulated secretory with reports that SP-C is an integral membrane protein (7Keller A. Eistetter H.R. Voss T. Schäfer K.P. Biochem. J. 1991; 277: 493-499Crossref PubMed Scopus (51) Google Scholar, A. W. Schäfer K.P. Voss T. Am. J. Respir. Cell Mol. Biol. PubMed Scopus Google Scholar), suggested that the proprotein was located in the limiting membrane of the and that to of SP-C the cell To this PC12 cells were transiently transfected with wild type and with an the N-terminal or C-terminal peptide domains and to the cell In SP-C was detected with the C-terminal peptide, with an for this peptide domain (Fig. The N-terminal propeptide was detected only after cell that this peptide domain is located in the (Fig. Collectively, these suggest that SP-C is a type II integral membrane protein and that sorting to the regulated secretory occurs of proprotein To the peptide sequence required for sorting of the SP-C proprotein to the regulated secretory a of deletion constructs (Fig. 2 were cloned in with transfected into PC12 and for with A. deletion a consisting of only the mature SP-C peptide with SP-C was sorted to the regulated secretory of the of at the N- or C of the mature SP-C peptide not two a at positions and (residues and of the proprotein) and two palmitoylated cysteines at positions 5 and 6 (residues 28 and 29 of the The sorting of the was by the 5 of the mature peptide the sorting of the palmitoylated cysteines was by both to or in the of the proprotein The proteins encoded by of these constructs with of PC12 that is in SP-C sorting not from these experiments that the N- and C-terminal peptide domains of SP-C are not required for sorting to the regulated secretory of PC12 cells and that a sorting is located in the 10-residue cytosolic domain of the mature peptide (residues and of the The results in PC12 cells that the mature SP-C peptide be sorted to lamellar bodies in type II This was by isolated mouse type II cells with a encoding the mature SP-C peptide The plasmid was and into type II cells with a h of culture, was detected in that also for the mature SP-B peptide (Fig. Since SP-B mature peptide is only detected in multivesicular bodies and lamellar bodies of type II cells W.F. T. Haagsman H.P. Weaver T.E. Whitsett J.A. van Golde Geuze H.J. Am. J. Physiol. Google Scholar), these results that the mature SP-C peptide is sorted to the secretory in the of the N- and C-terminal peptide domains. To SP-C mature peptide be sorted to lamellar an encoding SP-C mature peptide with a hemagglutinin was for intratracheal injection into mice. The of in mice was by of lung were detected throughout the regulated secretory the endoplasmic reticulum, Golgi, multivesicular bodies, and lamellar bodies, but not in the or membrane (Fig. B multivesicular bodies, was detected the limiting membrane and the (Fig. 4 to the of SP-C proprotein in wild type not and SP-B null mice (Fig. 4 of SP-C the of multivesicular bodies suggested that be To the cytosolic domain of the mature SP-C peptide (residues was sufficient to secretion of SP-C, mice were with mice were and the surfactant was was detected in the surfactant of with secretion of the peptide into the of into and that with wild type mature SP-B in the (Fig. 5 analyses of the from mice detected in epithelial cells and in the alveolar of mice were with adenovirus encoding was not detected in not indicate that SP-C mature peptide is secreted into the alveolar space and with the surfactant To be secreted in the of SP-C, SP-C mice were with after mice were and and were was detected in the of wild type but not SP-C null mice To was sorted to lamellar bodies in the of SP-C, lamellar bodies from lung were isolated from wild type and SP-C mice. was detected in lamellar body of wild type mice but not SP-C (−/−) (Fig. 6 from these experiments in the of SP-C, the cytosolic domain of the mature peptide is not sufficient to SP-C to the regulated secretory in type II To the that the N-terminal propeptide of SP-C sorting and secretion of in SP-C (−/−) an adenovirus encoding the SP-C N-terminal propeptide and mature peptide was after SP-C (−/−) mice were and the was detected in the surfactant that also mature SP-B peptide with in wild type with cleavage of the N-terminal propeptide. The results of these experiments indicate that the N-terminal propeptide is required for secretion of SP-C. To the cytosolic domain of SP-C sorting and secretion of a transmembrane domain, an consisting of the SP-C cytosolic domain (residues the platelet-derived growth factor receptor transmembrane domain (residues and the acid was after SP-C (−/−) mice were and the in lamellar body were isolated from lung was detected in both and lamellar body not with suggesting that the N-terminal propeptide was not from the results that the cytosolic domain of the SP-C proprotein the sorting and secretion of a transmembrane domain but is not sufficient to processing of the propeptide. SP-C is an integral membrane protein that is sorted to the lamellar body and secreted with surfactant lipids into the alveolar space. The study was to identify the peptide in the sorting of SP-C to the regulated secretory in type II cells and secretion of this integral membrane In the of SP-C, the mature peptide was sorted to the regulated secretory and was secreted into the airspace. in the of SP-C, the SP-C N-terminal propeptide was required for secretion of the mature peptide. The importance of the cytosolic domain for secretion of SP-C was by that of the proprotein was sufficient to secretion of a receptor. membrane proteins are sorted to specific information encoded in the cytosolic domain of the protein (14Hunziker W. Geuze H.J. Bioessays. 1996; 18: 379-389Crossref PubMed Scopus (237) Google Scholar, 15Marks M.S. Ohno H. Kirchhausen T. Bonifacino S.J. Trends Cell Biol. 1997; 7: 124-128Abstract Full Text PDF PubMed Scopus (277) Google Scholar). studies have the SP-C proprotein as a type III integral membrane protein (8Vorbroker D.K. Voorhout W.F. Weaver T.E. Whitsett J.A. Am. J. Physiol. 1995; 13: L727-L733Google Scholar), in which the C-terminal propeptide is located in the or a type II membrane integral membrane protein (7Keller A. Eistetter H.R. Voss T. Schäfer K.P. Biochem. J. 1991; 277: 493-499Crossref PubMed Scopus (51) Google Scholar), in which the N-terminal propeptide is The results of the study in PC12 cells suggest a type II membrane with the results by et al. (7Keller A. Eistetter H.R. Voss T. Schäfer K.P. Biochem. J. 1991; 277: 493-499Crossref PubMed Scopus (51) Google Scholar). This is also with the of a sequence and of the proprotein) and the of 28 and 29 of the that the cytosolic of the membrane J.T. 1998; PubMed Scopus Google G. J. Biochem. 1993; PubMed Scopus Google Scholar). from studies that detected the N-terminal propeptide the of multivesicular bodies, with a type II (8Vorbroker D.K. Voorhout W.F. Weaver T.E. Whitsett J.A. Am. J. Physiol. 1995; 13: L727-L733Google Scholar, 9Voorhout W.F. Weaver T.E. Haagsman H.P. Geuze H.J. van Golde L.M.J. Microsc. Res. Technique. 1993; 26: 366-373Crossref PubMed Scopus (67) Google Scholar). these results suggest that the cytosolic domain of SP-C is of the acid N-terminal propeptide and the first of the mature peptide. with a cytosolic for the N-terminal deletion of the acid C-terminal propeptide of the SP-C proprotein not sorting to the regulated secretory in PC12 of the 5 of the mature peptide (residues of the which contains a at positions and not SP-C SP-C was also sorted the deletion of the acid N-terminal suggesting that the acid cytosolic domain of the mature peptide (residues of the proprotein) is sufficient to SP-C to the regulated secretory pathway. This is to with results of studies suggesting that the C-terminal propeptide of the proprotein was critical for intracellular of SP-C A. W. Schäfer K.P. Voss T. Am. J. Respir. Cell Mol. Biol. PubMed Scopus Google Scholar, M.F. Lomax S.J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). It is that within the C-terminal peptide resulted in and of SP-C in a cytosolic for in a was by deletion of that are in the SP-C proprotein of species to A. W. Schäfer K.P. Voss T. Am. J. Respir. Cell Mol. Biol. PubMed Scopus Google Scholar, M.F. Lomax S.J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S.J. Lomax M.F. Am. J. Physiol. 277: PubMed Google Scholar). of the studies is by of SP-C deletion constructs in cell that a regulated secretory pathway. PC12 cells in this study have a regulated secretory and have for sorting and secretion studies K. S. Whitsett J.A. Res. PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar, J. Res. 1996; PubMed Scopus Google Scholar, A.D. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, T. S. K. B. T. D. G. Jornvall H. S. FEBS Lett. PubMed Scopus Google Scholar). have previously PC12 cells to identify a sorting in the SP-B proprotein and the of the sorting in mice (16Lin S. Akinbi H.T. Breslin J.S. Weaver T.E. J. Biol. Chem. 1996; 271: 19689-19695Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, S. Weaver T.E. BBA Mol. Cell Res. 1996; PubMed Scopus Google Scholar). In the the results of experiments in PC12 cells were by isolated type II cells and by intratracheal injection of wild type mice with adenovirus encoding the mature peptide. In both SP-C mature peptide was detected in lamellar bodies, with sorting of the mature peptide to this compartment in the of the N- and C-terminal peptide domains. SP-C was not detected in lamellar bodies or of SP-C (−/−) mice with adenovirus encoding This suggests that in wild type mice SP-C with transfected mature SP-C peptide to its to lamellar SP-C mature peptide with an in PC12 leading to in secretory The sorting in SP-C (−/−) mice was by the addition of the acid N-terminal propeptide to was to the mature peptide with the multivesicular body, a compartment previously to be in processing of the SP-C and SP-B (8Vorbroker D.K. Voorhout W.F. Weaver T.E. Whitsett J.A. Am. J. Physiol. 1995; 13: L727-L733Google Scholar, 10Beers M.F. Lomax C. Am. J. Physiol. 1995; 13: L744-L753Google Scholar, W.F. T. Haagsman H.P. Weaver T.E. Whitsett J.A. van Golde Geuze H.J. Am. J. Physiol. Google the peptide was detected in the of Collectively, these studies suggest that the N-terminal propeptide is required for sorting of SP-C to the secretory in type II cells and that sorting occurs of the C-terminal peptide. the cytosolic domain of SP-C was to both the sorting lamellar and secretion of the transmembrane domain of was not to a suggesting that the SP-C transmembrane domain to the formation of an cleavage the propeptide and mature peptide. Sorting of SP-C to the secretory is but not sufficient for secretion of SP-C into the alveolar space. In transfected PC12 SP-C was detected in the limiting membrane of granules and was to the membrane in type II epithelial SP-C was detected in the lumen of the lamellar body and was secreted with surfactant suggest that the SP-C proprotein is from the limiting membrane of a or storage to the lumen in the secretory of the type II cell. analyses detected SP-C proprotein in the limiting membrane and luminal of multivesicular bodies, suggesting a role for this compartment in SP-C of SP-C within the multivesicular body is probably the of of the limiting to the leading to internalization and of the growth factor receptor in lysosomes S. K. G. A. J. 1990; Full Text PDF PubMed Scopus Google Scholar, A. J. Cell Biol. 1996; PubMed Scopus Google Scholar, T. A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). of the multivesicular membrane is probably dependent the of specific in the cytosolic domain of the SP-C proprotein with proteins the limiting membrane of the multivesicular body are in the K. B. A. K. R. Biochem. J. 1997; PubMed Scopus Google Scholar, W. Geuze H.J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), suggesting that SP-C is sorted to the The that is secreted of SP-C (−/−) mice suggests that of the proprotein of that sorting to the secretory and internalization of SP-C into the luminal of the multivesicular bodies. the multivesicular body fuses with the lamellar body, in the of the of the multivesicular body into the of the lamellar body secretion of SP-C with surfactant phospholipids Whitsett J.A. Weaver T.E. PubMed Scopus Google Scholar, J. Scopus Google Scholar). In the mature SP-C peptide is sorted to the regulated secretory of PC12 isolated mouse type II and wild type mature SP-C peptide was not sorted to lamellar bodies or secreted into the of SP-C (−/−) mice. The addition of the SP-C N-terminal propeptide to the mature peptide restored and secretion of SP-C as of a surfactant the cytosolic domain of SP-C was sufficient to the sorting and secretion of a transmembrane domain. studies that the N-terminal propeptide of SP-C is critical for the intracellular and secretion of SP-C in type II