Post-Golgi Vesicles Cotransport Docosahexaenoyl-Phospholipids and Rhodopsin during Frog Photoreceptor Membrane Biogenesis

Post-Golgi vesicles budding from the trans-Golgi network (TGN) are involved in the vectorial transport and delivery of rhodopsin to photoreceptor rod outer segments (ROS). We report here that newly synthesized docosahexaenoyl (DHA) phospholipids are sequestered and cotransported by rhodopsin-bearing post-Golgi vesicles to ROS. Frog retinas were pulse-labeled with [35S]methionine/cysteine and [3H]DHA prior to ROS isolation and subcellular fractionation. After a 1-h pulse, relatively uniform [3H]DHA-lipid labeling (DPM/μg protein) was observed in all fractions enriched in post-Golgi vesicles, TGN, Golgi, and endoplasmic reticulum (ER) membranes. During the subsequent 2-h chase translocation of free [3H]DHA from ROS to the photoreceptor inner segment contributed to an additional overall increase in labeling of lipids. The specific activity (dpm/nmol DHA) in ER-enriched fraction was similar or higher than in other subcellular fractions after both the pulse and the chase, indicating that the bulk of [3H]DHA-lipids was synthesized in the ER. After the chase a 2-fold increase in labeling of lipids in the ER and Golgi and a 2.6-fold in lighter TGN-enriched fractions was observed. The highest labeling was in the post-Golgi vesicle fraction (4-fold increase), with [3H]DHA-phosphatidylcholine and [3H]DHA-phosphatidylethanolamine showing the greatest increase. At the same time, newly synthesized [35S]rhodopsin shifted from the ER and Golgi toward TGN and post-Golgi fractions. Therefore, sequestration and association of [35S]rhodopsin and [3H]DHA-lipids in a TGN membrane domain occurs prior to their exit and subsequent vectorial cotransport on post-Golgi vesicles to ROS. Labeling of ROS lipids was very low, with phosphatidylinositol and diacylglycerols displaying the highest labeling. This indicates that other mechanisms by-passing Golgi, i.e. facilitated by lipid carrier proteins, may also contribute to molecular replacement of disc membrane DHA-phospholipids, particularly phosphatidylinositol. Post-Golgi vesicles budding from the trans-Golgi network (TGN) are involved in the vectorial transport and delivery of rhodopsin to photoreceptor rod outer segments (ROS). We report here that newly synthesized docosahexaenoyl (DHA) phospholipids are sequestered and cotransported by rhodopsin-bearing post-Golgi vesicles to ROS. Frog retinas were pulse-labeled with [35S]methionine/cysteine and [3H]DHA prior to ROS isolation and subcellular fractionation. After a 1-h pulse, relatively uniform [3H]DHA-lipid labeling (DPM/μg protein) was observed in all fractions enriched in post-Golgi vesicles, TGN, Golgi, and endoplasmic reticulum (ER) membranes. During the subsequent 2-h chase translocation of free [3H]DHA from ROS to the photoreceptor inner segment contributed to an additional overall increase in labeling of lipids. The specific activity (dpm/nmol DHA) in ER-enriched fraction was similar or higher than in other subcellular fractions after both the pulse and the chase, indicating that the bulk of [3H]DHA-lipids was synthesized in the ER. After the chase a 2-fold increase in labeling of lipids in the ER and Golgi and a 2.6-fold in lighter TGN-enriched fractions was observed. The highest labeling was in the post-Golgi vesicle fraction (4-fold increase), with [3H]DHA-phosphatidylcholine and [3H]DHA-phosphatidylethanolamine showing the greatest increase. At the same time, newly synthesized [35S]rhodopsin shifted from the ER and Golgi toward TGN and post-Golgi fractions. Therefore, sequestration and association of [35S]rhodopsin and [3H]DHA-lipids in a TGN membrane domain occurs prior to their exit and subsequent vectorial cotransport on post-Golgi vesicles to ROS. Labeling of ROS lipids was very low, with phosphatidylinositol and diacylglycerols displaying the highest labeling. This indicates that other mechanisms by-passing Golgi, i.e. facilitated by lipid carrier proteins, may also contribute to molecular replacement of disc membrane DHA-phospholipids, particularly phosphatidylinositol. Vertebrate photoreceptors are polarized and compartmentalized cells, with a photosensitive outer segment and a synaptic terminal domain at opposite ends of the cell. These domains are attached through a connecting cilium and a short axon, respectively, to a central region, the inner segment. The inner segment is the site where lipid and protein synthesis, polarized sorting of molecules, and initiation of membrane biogenesis for both the outer segment and synaptic terminals take place. Disc membranes in rod outer segments (ROS) 1The abbreviations used are: ROS, rod outer segment(s); PLs, phospholipids; DHA, 22:6n-3, docosahexaenoic acid; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; DAG, diacylglycerol; ER, endoplasmic reticulum; TGN, trans-Golgi network; TP, transfer proteins. 1The abbreviations used are: ROS, rod outer segment(s); PLs, phospholipids; DHA, 22:6n-3, docosahexaenoic acid; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; DAG, diacylglycerol; ER, endoplasmic reticulum; TGN, trans-Golgi network; TP, transfer proteins. display a unique lipid-protein composition. The visual pigment rhodopsin, which accounts for more than 85% of disc membrane proteins (1Papermaster D.S. Dreyer W.J. Biochemistry. 1974; 13: 2438-2444Crossref PubMed Scopus (574) Google Scholar), is embedded within a highly fluid lipid bilayer comprised of phospholipids (PLs) highly enriched in docosahexaenoic acid (DHA, 22:6n-3) (2Aveldaño de Caldironi M.I. Bazan N.G. Neurochem. Int. 1980; 1: 381-392Crossref Scopus (30) Google Scholar, 3Aveldaño M.I. Bazan N.G. J. Lipid Res. 1983; 24: 620-627Abstract Full Text PDF PubMed Google Scholar, 4Fliesler S.J. Anderson R.E. Prog. Lipid Res. 1983; 22: 79-131Crossref PubMed Scopus (791) Google Scholar, 5Wiegand R.D. Anderson R.E. Exp. Eye Res. 1983; 37: 150-173Crossref Scopus (49) Google Scholar, 6Choe H.-G. Anderson R.E. Exp. Eye Res. 1990; 51: 159-165Crossref PubMed Scopus (29) Google Scholar). Amphibian photoreceptor cells are a useful experimental model to study protein and lipid trafficking in a polarized cell. Photoreceptors actively synthesize proteins (mainly rhodopsin) and DHA-PLs to support the dynamic daily renewal of 50–80 large disc membranes in each rod cell that results in the addition of membrane at ≃3 μm2/min (7Besharse J.C. The Retina: A Model for Cell Biological Studies.in: Adler R. Farber D. Academic Press, Inc., New York1986: 297-352Google Scholar, 8Deretic D. Papermaster D.S. Progress in Retinal and Eye Research.in: Osborne N.N. Chadek G.J. 14. Pergamon Press Inc., Tarrytown, NY1995: 249-265Google Scholar). ROS lack the capacity for de novo synthesis of PLs (4Fliesler S.J. Anderson R.E. Prog. Lipid Res. 1983; 22: 79-131Crossref PubMed Scopus (791) Google Scholar, 9Choe H.-G. Ghalayini A.J. Anderson R.E. Exp. Eye Res. 1990; 51: 167-176Crossref PubMed Scopus (21) Google Scholar). Therefore, they depend entirely on an external supply of PLs from the inner segment where they are synthesized mainly in the rough endoplasmic reticulum (ER) (10Mercurio A.M. Holtzman E. J. Neurocytol. 1982; 11: 295-322Crossref PubMed Scopus (24) Google Scholar). How these highly unsaturated lipids become components of ROS membranes and at which stage of membrane biosynthesis and disc morphogenesis they become associated with rhodopsin is not yet clear. Newly synthesized rhodopsin is vectorially transported from its site of synthesis in the rough ER to ROS by vesicles that bud from the trans-Golgi network (TGN), cluster beneath the connecting cilium, and fuse with the inner segment plasma membrane within the periciliary ridge complex (11Papermaster D.S. Schneider B.G. Defoe D. Besharse J.C. J. Histochem. Cytochem. 1986; 34: 5-16Crossref PubMed Scopus (63) Google Scholar, 12Deretic D. Papermaster D.S. J. Cell Biol. 1991; 113: 1281-1293Crossref PubMed Scopus (106) Google Scholar). A very low buoyant density (ρ = 1.09 g/ml) post-Golgi vesicular subcellular fraction carrying newly synthesized rhodopsin has been isolated and characterized from frog retinal photoreceptor cells (12Deretic D. Papermaster D.S. J. Cell Biol. 1991; 113: 1281-1293Crossref PubMed Scopus (106) Google Scholar, 13Deretic D. Papermaster D.S. J. Cell Sci. 1993; 106: 803-813PubMed Google Scholar, 14Deretic D. Aebersold R.H. Morrison H.D. Papermaster D.S. J. Biol. Chem. 1994; 269: 16853-16861Abstract Full Text PDF PubMed Google Scholar, 15Deretic D. Huber L.A. Ransom N. Mancini M. Simons K. Papermaster D.S. J. Cell Sci. 1995; 108: 215-224Crossref PubMed Google Scholar). Whereas rhodopsin and the bulk of DHA-PLs are synthesized in the rough ER and both can follow a vesicle-mediated traffic through the biosynthetic pathway, other mechanisms can contribute to trafficking and selective delivery of PLs to intracellular organelles (16Bishop W.R. Bell R.M. Annu. Rev. 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Cell Biol. 1991; 113: 1281-1293Crossref PubMed Scopus (106) Google Scholar, D. Papermaster D.S. for the of Press, New Scholar). [3H]DHA was is actively PLs in the inner segment of photoreceptor K. R.D. Anderson R.E. Biochemistry. 1988; PubMed Scopus Google Scholar, N.G. in the L.A. Press, Scholar, N.G. Bazan Prog. Lipid Res. 1986; PubMed Scopus Google Scholar, M.I. PubMed Scopus (24) Google Scholar, M.I. PubMed Scopus Google prior to their vectorial transport to the at the of ROS and also to synaptic terminals de Bazan N.G. J. 1991; 11: PubMed Google de Bazan N.G. Curr. Eye Res. 1993; 13: Scopus Google Scholar). results that newly synthesized the components of disc membranes and are and with newly synthesized [35S]rhodopsin in post-Golgi i.e. and are very synthesized and to ROS by that the Golgi and may facilitated by carrier proteins. were from in a and for a prior to the activity and protein labeling were from were from and acid and were from were from other used were of the highest from frog the of were and of retinas were in of an D. Aebersold R.H. Morrison H.D. Papermaster D.S. J. Biol. Chem. 1994; 269: 16853-16861Abstract Full Text PDF PubMed Google Scholar, N.G. in the L.A. Press, at in the of [3H]DHA and protein labeling for of retinas was for in prior to subcellular fractionation. the in of [3H]DHA is at de Bazan N.G. J. 1991; 11: PubMed Google Scholar, de Bazan N.G. Curr. Eye Res. 1993; 13: Scopus Google Scholar, de N. N.G. Scholar), was not the Therefore, chase to the for the isolation of ROS and subcellular been in (12Deretic D. Papermaster D.S. J. Cell Biol. 1991; 113: 1281-1293Crossref PubMed Scopus (106) Google Scholar, D. Papermaster D.S. for the of Press, New Scholar). the pulse and retinas were through a and ROS were by on Retinal were in in and for at Inc., The after is enriched in photoreceptor biosynthetic membranes and organelles involved in rhodopsin transport (12Deretic D. Papermaster D.S. J. Cell Biol. 1991; 113: 1281-1293Crossref PubMed Scopus (106) Google Scholar, D. Papermaster D.S. for the of Press, New Scholar). post-Golgi vesicles from TGN, Golgi, and ER the was on a in and a of in the same were for at in a at fractions were from the of the A was used to the and to the fractions. The subcellular fractions were with and at for in a The were in of and for protein and for lipid was (12Deretic D. Papermaster D.S. J. Cell Biol. 1991; 113: 1281-1293Crossref PubMed Scopus (106) Google Scholar). rhodopsin was in subcellular fractions by of to and the of associated with the rhodopsin was and by a proteins were to PubMed Scopus Google Scholar), a were from the fractions by of the J. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). phospholipids and lipids were isolated in the same a H.-G. Anderson R.E. Exp. Eye Res. 1990; 51: 159-165Crossref PubMed Scopus (29) Google an of the lipid and lipid a was on the from each of with and for at at The was in the the the from the of the After with the was to the and lipid and were from the and from the were in to lipids that at the of the The was with a to lipid from of the from the and the was from the prior to the in the This in the same the the isolation of and free that with the Lipid were by and the was in a of lipid were for of acid and composition. acid were in by with of acid for at after the with and with a The were at and of of and a of and were acid in were a inner by a carrier in a The and were and respectively, and the was from to Bazan N.G. J. 1990; 1: Scopus Google Scholar). The were by by with the of acid and the for [3H]DHA lipid labeling and [35S]rhodopsin are a for = were for of was newly synthesized and [35S]rhodopsin are transported in the same of post-Golgi vesicles in fraction of the retinas were pulse-labeled for in the of both and for in prior to subcellular fractionation. This experimental labeling of newly synthesized rhodopsin within 1-h pulse and a labeling of the vesicles the 2-h chase (12Deretic D. Papermaster D.S. J. Cell Biol. 1991; 113: 1281-1293Crossref PubMed Scopus (106) Google Scholar, D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the [35S]rhodopsin labeling from the fractions was similar for pulse and chase not [3H]DHA was by from retinas after a 1-h pulse to retinas after a 2-h After of pulse of [35S]rhodopsin was in TGN and Golgi (12Deretic D. Papermaster D.S. J. Cell Biol. 1991; 113: 1281-1293Crossref PubMed Scopus (106) Google Scholar). After a 2-h a toward post-Golgi fractions was with fraction displaying the greatest increase The of [3H]DHA subcellular fractions was similar to that of [35S]rhodopsin with the highest observed in fractions that also the of membranes after the 2-h chase post-Golgi fraction higher [3H]DHA labeling at the newly synthesized [35S]rhodopsin in labeling in fractions and was than the Labeling from subcellular fractions protein is in and after a 1-h pulse, all fractions similar labeling of [3H]DHA after the subsequent 2-h labeling of at 2-fold in the fractions that to the density of ER D. Papermaster D.S. J. Cell Sci. 1993; 106: 803-813PubMed Google Scholar, 14Deretic D. Aebersold R.H. Morrison H.D. Papermaster D.S. J. Biol. Chem. 1994; 269: 16853-16861Abstract Full Text PDF PubMed Google Scholar), Golgi (11Papermaster D.S. Schneider B.G. Defoe D. Besharse J.C. J. Histochem. Cytochem. 1986; 34: 5-16Crossref PubMed Scopus (63) Google Scholar, 12Deretic D. Papermaster D.S. J. Cell Biol. 1991; 113: 1281-1293Crossref PubMed Scopus (106) Google Scholar), and TGN (10Mercurio A.M. Holtzman E. J. Neurocytol. 1982; 11: 295-322Crossref PubMed Scopus (24) Google Scholar), by 2.6-fold in lighter TGN fractions and at higher labeling in post-Golgi vesicles in fraction with pulse-labeled pulse and chase labeling was observed in the free [3H]DHA indicating an the of the to these membrane and its lipids by de novo synthesis The of labeling observed in fraction may the of free [3H]DHA contributed by a of ROS fractions and at a buoyant density of (12Deretic D. Papermaster D.S. J. Cell Biol. 1991; 113: 1281-1293Crossref PubMed Scopus (106) Google Scholar). of the from ROS after pulse and chase labeling was free and ROS lipids labeling was the all subcellular fractions A increase in ROS [3H]DHA-lipids labeling after the to protein) for by the of of free [3H]DHA to The specific activity of [3H]DHA-lipids (dpm/nmol of that fraction (ER) after both the pulse DHA) and the DHA) similar or higher than other fractions. After the pulse the specific activity of fraction DHA) was than that of fraction After the chase, the specific activity of fraction DHA) was not from the specific activity of fraction The PLs in retinal and PE, a similar labeling after a 2-h and from fractions enriched in ER to fractions of the TGN, with a in post-Golgi fraction a and respectively, pulse labeling. The of was very similar to that of and their labeling was in TGN fractions with the highest increase in post-Golgi fraction and was the that not a of labeling in fraction increase fractions and acid labeling from ER fractions to post-Golgi fraction with from the 1-h pulse labeling. labeling in ROS was very low with a of than all other subcellular fractions the of ROS that is in the fraction and may contribute to their highly free [3H]DHA labeling contribute to results in an of lipid labeling in these fractions that from inner segment membranes. by the highest labeling in ROS, with a increase after a 2-h by PC, and were observed a 1-h pulse and 2-h labeling in and This may the labeling of disc membrane lipids by molecular replacement [3H]DHA by the transport from the inner segment of a of newly synthesized [3H]DHA-lipids of newly ROS of to (4Fliesler S.J. Anderson R.E. Prog. Lipid Res. 1983; 22: 79-131Crossref PubMed Scopus (791) Google Scholar, Exp. Eye Res. 1995; PubMed Scopus (21) Google Scholar). The of [3H]DHA for PI, PC, and in all fractions for ROS fraction they a similar in other labeling The highest labeling of was observed in fraction and to a in fraction associated with proteins at the of the that may after the (12Deretic D. Papermaster D.S. J. Cell Biol. 1991; 113: 1281-1293Crossref PubMed Scopus (106) Google Scholar). The to labeling was higher in TGN fractions with post-Golgi fractions showing the highest in fraction of the TGN after both pulse and chase labeling mainly membrane from the ER-enriched fractions protein) to the post-Golgi vesicular fractions protein) The was very similar for all fractions of for TGN fractions where ROS, not prior to subcellular ROS, for of The in fraction with ROS that the lipids from the vesicles rhodopsin are enriched in that lipids contributed by other vesicles with a of are in The is of rhodopsin-bearing post-Golgi vesicles with that they of the vesicles in fraction of the (12Deretic D. Papermaster D.S. J. Cell Biol. 1991; 113: 1281-1293Crossref PubMed Scopus (106) Google Scholar). The of protein in ROS was that of lipids from fraction vesicles rhodopsin is the protein in fraction its to the proteins in the fraction is than rhodopsin accounts for of ROS proteins (12Deretic D. Papermaster D.S. J. Cell Biol. 1991; 113: 1281-1293Crossref PubMed Scopus (106) Google Scholar). Therefore, the of DHA-PLs with to rhodopsin protein in these post-Golgi vesicles may similar to that of ROS. This study the the and association of newly synthesized with [35S]rhodopsin in frog photoreceptors the membrane components their from the rough ER, where they are through the Golgi, and the TGN on post-Golgi vesicles vectorially to ROS for the of disc membranes. results also other free [3H]DHA may by in disc PLs than in the inner segment for mainly are actively synthesized in the inner transported to ROS, and disc the short of in labeling the labeling of all disc membranes by molecular replacement transport of [3H]DHA by with the labeling of a at the by membrane to the of the to the overall labeling of ROS lipids. The of [35S]rhodopsin labeling through the of the its vesicle-mediated vectorial traffic from the site of synthesis at the rough ER to ROS. The of [35S]rhodopsin in Golgi fractions by of labeling by its the subsequent 2-h chase toward TGN and post-Golgi vesicles in fraction and was used a of membrane to follow the of newly synthesized lipids in Golgi and TGN-enriched fractions at newly synthesized lipids that become components of the membranes the [3H]DHA-lipid that are in through these to [3H]DHA-lipids labeling of post-Golgi vesicles is a of newly synthesized [3H]DHA-lipids cotransported with [35S]rhodopsin to ROS. the of pulse [3H]DHA lipids similar in all subcellular fractions. This [3H]DHA-lipids all fractions was also in a similar of fractions with ROS higher newly synthesized [3H]DHA-lipids were transported the of the biosynthetic by vesicle budding and by carrier proteins, by through R.E. Curr. Opin. Cell Biol. 1990; 2: 652-663Crossref PubMed Scopus (81) Google Scholar, 18Voelker D.R. Rev. 1991; PubMed Google Scholar). of [3H]DHA by in lipids trafficking the transport also contribute to the uniform labeling observed subcellular fractions. After the 2-h labeling in that the labeling of free [3H]DHA in ROS was not by an disc membrane phospholipids by a translocation to the inner segment where was actively This by the of acid proteins in ROS 1993; 34: and in the fraction of retinas Sci. 1994; Google Scholar, J. Sci. 1995; Google and in This that prior to its lipids the highest activity in from frog retinas and very low activity in ROS Bazan N.G. Curr. Eye Res. PubMed Scopus Google Scholar). free can in disc membrane PLs by of their M.I. M.I. 1986; PubMed Scopus Google Scholar, Exp. Eye Res. 1988; PubMed Scopus Google Scholar, K. Anderson R.E. Exp. 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Cell Biol. 1983; PubMed Scopus (29) Google and in at the of the ROS study that newly synthesized mainly and are vectorially cotransported to ROS by rhodopsin-bearing post-Golgi vesicles, and other PLs may also ROS and the TGN by-passing the Golgi by transfer proteins. in the complex of membrane addition of rhodopsin and DHA-PLs at the of ROS for the of other PLs that not of PLs in disc the periciliary by to the that contribute to the complex polarized trafficking of DHA-PLs by vesicular by transfer transport to ROS. We R. E. Anderson for of the results and and for their