(Received for publication, July 19, 1995; and in revised form, October 2, 1995)
From the
In retinal photoreceptors, highly polarized organization of the light-sensitive organelle, the rod outer segment, is maintained by the sorting of rhodopsin and its associated proteins into distinct post-Golgi vesicles that bud from the trans-Golgi network (TGN) and by their vectorial transport toward the rod outer segment. We have developed an assay that reconstitutes the formation of these vesicles in a retinal cell-free system. Vesicle formation in this cell-free assay is ATP-, GTP-, and cytosol-dependent. In frog retinas vesicle budding also proceeds at 0 °C, both in vivo and in vitro. Vesicles formed in vitro are indistinguishable from the vesicles formed in vivo by their buoyant density, protein composition, topology, and morphology. In addition to the previously identified G-proteins, these vesicles also contain rab11. Concurrently with vesicle budding, resident proteins are retained in the TGN. Collectively these data suggest that rhodopsin and its associated proteins are sorted upon exit from the TGN in this cell-free system. Removal of membrane-bound GTP-binding proteins of the rab family by rab GDP dissociation inhibitor completely abolishes formation of these vesicles and results in the retention of rhodopsin in the Golgi. A monoclonal antibody to the cytoplasmic (carboxyl-terminal) domain of rhodopsin and its Fab fragments strongly inhibit vesicle formation and arrest newly synthesized rhodopsin in the TGN rather than the Golgi. Therefore rhodopsin sorting at the exit from the TGN is mediated by the interaction of its cytoplasmic domain with the intracellular sorting machinery.
Reconstitution of intracellular sorting events in cell-free
systems has significantly contributed to our understanding of the
molecular mechanisms that underlie membrane trafficking. Together with
genetic studies in yeast and studies of regulated exocytosis of
synaptic vesicles, in vitro assays reconstituting intra-Golgi
transport have yielded invaluable information about common mechanisms
involved in membrane budding and fusion from yeast to mammals (for
review see Rothman and Warren(1994) and Rothman(1994)). Several
cell-free assays have been employed to reconstitute early steps in
exocytosis (endoplasmic reticulum to Golgi and intra-Golgi transport)
and endocytosis. A limited number of studies have established in
vitro conditions for cell-free post-Golgi vesicle formation from
the TGN ()(de Curtis and Simons, 1989; Tooze and Huttner,
1990; Salamero et al., 1990; Ohashi and Huttner, 1994).
However, these events have not been studied in the primary tissues of
neuronal origin.
Retinal photoreceptors are highly polarized sensory
neurons with three distinct plasma membrane domains: rod outer segments
(ROS) that capture light, the lateral plasma membrane of the inner
segment, and the axon with the synaptic terminal. Photoreceptors are
excellent models for cell biological studies because continuous ROS
membrane renewal by polarized sorting of rhodopsin and its associated
proteins on post-Golgi vesicles results in the addition of up to 3
µm
/min of ROS membranes (reviewed in Besharse(1986),
Simons and Zerial(1993), and Deretic and Papermaster(1995)).
Rhodopsin-bearing post-Golgi vesicles isolated from frog retinas can be
distinguished from all other subcellular compartments by several
criteria, including kinetics of formation, buoyant density, and protein
composition (Deretic and Papermaster, 1991). Several proteins of the
small G-protein rab family and
-crystallins are vesicle-associated
proteins and potential regulators of vesicle budding and sorting
(Deretic and Papermaster, 1993a, 1995; Deretic et al., 1994,
1995). However, the mechanism of action of these associated proteins is
still elusive in the absence of an assay that allows access to the
intracellular compartments involved in vesicle formation.
Inherited retinal diseases in humans and in animal models suggest that some of the mutations in the rhodopsin gene may lead to transport defects that ultimately result in retinal degeneration and blindness (Dryja et al., 1990; Olsson et al., 1992; Roof et al., 1994; Sung et al., 1991, 1994). The rhodopsin residues that are mutated in inherited retinal degenerations are conserved in the frog rhodopsin sequence, consistent with their important contribution to the stability of rhodopsin structure and its function in the photoreceptor cell (Pittler et al., 1992). In addition, retinal cells also appear to be very sensitive to the defects in isoprenylation of small G-proteins and rab proteins in particular (Seabra et al., 1993; Pittler et al., 1995). Therefore, identification of the proteins that interact with rhodopsin and guide it on its journey to the ROS, as well as the establishment of the role of rab proteins in these processes are of essential importance for the understanding of the establishment and maintenance of photoreceptor polarity and health.
We have developed a cell-free assay that reconstitutes not only rhodopsin transport through the Golgi but also post-Golgi vesicle budding. Due to the complexity of the retinal tissue, biosynthetic organelles of the rod inner segments are accessible only upon removal of the ROS and the neural retina. Using the fractionation technique described before (Deretic and Papermaster, 1991), we have generated a frog retinal PNS that is highly enriched in the photoreceptor cell's biosynthetic membranes. When supplemented with ATP and an exogenous ATP-regenerating system, this PNS is able to support rhodopsin transport and sorting in vitro. We used this cell-free assay to study the factors involved in the biogenesis of rhodopsin-bearing post-Golgi vesicles. We find that vesicle formation depends on the access of cytoplasmic proteins to the carboxyl-terminal domain of rhodopsin in the TGN membranes and on the presence of membrane-bound rab proteins.
Southern leopard frogs, Rana berlandieri (100-250 g) purchased from the Rana Co. (Brownsville, TX)
were maintained in a 12-h light/dark cycle and fed live crickets.
[S]-Express protein labeling mixture (1000
Ci/mmol), [
P]GTP (3000 Ci/mmol), and
[
H]-CMP-N-acetyl-neuraminic acid
(5-35 Ci/mmol) were from DuPont NEN, thermolysin was from
Calbiochem-Behring Corp. (La Jolla, CA), and ATP, GTP
S, creatine
phosphate, creatine phosphokinase (800 units/mg), and hexokinase (450
units/mg) were from Boehringer Mannheim. Ampholines were from Pharmacia
Biotech Inc., peroxidase-conjugated anti-rabbit IgG was from Kirkegaard
and Perry (Gaithersburg, MD), and the ECL Western blotting Detection
System and Hyperfilm ECL were from Amersham Corp. Several antibodies
and reagents were kindly provided for this study: rabbit antiserum and
affinity-purified antibody to the human rab6 expressed in Escherichia coli and recombinant bovine His
-tagged
rab GDI by Dr. Bruno Goud (Institut Pasteur, Paris) and anti-rab11
antiserum by Dr. Robert Parton (EMBL, Heidelberg).
To obtain
PNS enriched in photoreceptor biosynthetic membranes, retinal
fractionation was performed as described by Deretic and Papermaster
(1991, 1993b). Retinas were sheared through a 14-gauge needle, and ROS
were removed by flotation on 34% sucrose. Retinal pellets were
rehomogenized in 0.25 M sucrose in 10 mM Tris
acetate, pH 7.4, containing 1 mM MgCl and spun at
4000 rpm (1250 g
) (JA20 rotor, Beckman
Instruments, Inc., Palo Alto, CA) for 4 min. Supernatant obtained after
this centrifugation (PNS) contains biosynthetic organelles that
participate in rhodopsin transport (Papermaster et al., 1975;
Deretic and Papermaster, 1991), and it was used in subsequent in
vitro incubations.
In some experiments PNS was subfractionated into
membrane-enriched and cytosolic fractions. 1 ml of PNS in 0.25 M sucrose (obtained from 14 retinas) was overlaid on a step sucrose
gradient containing 1 ml of 0.5 M sucrose and 3 ml of 49%
sucrose. Gradients were spun for 1 h at 28,000 rpm (75,000 g) in an SW 50.1 rotor (Beckman). Soluble
proteins that remained in 0.25 M sucrose were further
centrifuged at 75,000 rpm (230,000 g
) in an TLA
100.3 rotor (Beckman) to remove residual membranes, and supernatants
containing cytosolic proteins were collected. Membrane proteins that
entered the 0.5 M sucrose layer were collected and divided in
half. To one half of the membranes 0.5 ml of 5 mg/ml bovine serum
albumin in 0.25 M sucrose was added (-cytosol), and to
the other half 0.5 ml of cytosolic proteins was added (+cytosol),
restoring photoreceptor-enriched PNS to the original ratio of
cytosol:membranes. Reconstituted PNS (±cytosol) was assayed for
vesicle formation after addition of 10
buffer stock, ATP, and an
ATP-regenerating system as described above.
Upon completion of
cell-free vesicle formation, assay mixtures were overlaid on 10
ml-linear 20-39% (w/w) sucrose gradients in 10 mM Tris
acetate, pH 7.4, and 1 mM MgCl above a 0.5-ml
cushion of 49% (w/w) sucrose. After centrifugation at 28,000 rpm
(100,000 g
) in an SW 40 rotor (Beckman) for 15 h
at 4 °C, 0.9-ml fractions were collected from the top of the
gradient. For each fraction the refractive index was determined from an
aliquot, and the remainder was diluted with 10 mM Tris
acetate, pH 7.4, and centrifuged at 50,000 rpm (230,000 g
) for 40 min in a SW 50.1 rotor or at 75,000 rpm
in an TLA 100.3 rotor. In some experiments subcellular fractions were
pooled according to the kinetics of their acquisition of radiolabeled
rhodopsin. Pellets were resuspended in 10 mM Tris acetate, pH
7.4, and aliquoted for analysis by SDS-PAGE or two-dimensional gel
electrophoresis.
Immunoblotting was performed as described (Deretic et
al., 1995) using the ECL Western blotting Detection System
(Amersham Corp.). GTP-binding proteins were detected by
[P]GTP overlays as described (Deretic and
Papermaster, 1993a). Blots were autoradiographed at -70°C
using Kodak BioMax MR film with intensifying screens. Autoradiograms
and Hyperfilm ECL were scanned using the Image processing and analysis
program (Wayne Rasband, NIH).
Figure 1: Rhodopsin-bearing post-Golgi vesicles are formed in the cell-free assay in the presence of ATP. Top panel, frog retinas are pulse-labeled for 60 min and then chased for 2 h either as a whole retina (in vivo, 1) or in the cell-free assay (in vitro) supplemented with ATP and an ATP-regenerating (+ATP, 2) or with an ATP-depleting system (-ATP, 3) at 22 °C. After retinal subcellular fractionation, radiolabeled proteins from two frog retinas are separated by SDS-PAGE and autoradiographed. The rhodopsin region of the autoradiograms is shown. Bottom panel, densitometric scans of the three autoradiograms. The distribution of newly synthesized rhodopsin in retinal subcellular fractions is expressed as a percentage of the total radiolabeled rhodopsin. This experiment was repeated six (1), eight (2), or three (3) times with similar results (see Fig. 4).
Figure 4:
Cytosol and G-proteins are required for
vesicle formation. Retinal subcellular fractions 4-6, which are
enriched in the post-Golgi vesicles, contain 16% of newly synthesized
rhodopsin after a 60-min pulse (1) and 32% following the 2 h
chase in vivo at 22 °C (2). In the presence of
ATP in the cell-free chase, a similar amount of radiolabeled rhodopsin
is found in these fractions at 22 (5) and 0 °C (7). ATP depletion completely inhibits additional vesicle
formation during the chase at both 22 (4) and 0 °C (6), i.e. there is no difference from the
distribution during the pulse. Upon cytosol removal, vesicle formation
during the chase is abolished by 75% (9), and when cytosol is
added back it is completely restored (10). 1 µM GTPS diminishes vesicle formation to
50% of control (8). The data are expressed as the mean ± S.E. of three
to eight separate experiments.
To study the role of intrinsic and
associated components that regulate vesicle formation, we have
reconstituted this process in a cell-free system. For this purpose we
modified the assay described by Tooze and Huttner(1990, 1992). Retinal
proteins are pulse-labeled for 60 min in the isolated frog retinas, the
rod outer segments are removed, and the remainders of the retinas are
homogenized. After low speed centrifugation to remove neural retina and
nuclei, retinal PNS is supplemented with ATP and an ATP-regenerating
system or with an ATP-depleting system (Davey et al., 1985)
and incubated at 22 °C for an additional 2 h. Fig. 1shows
that in PNS supplied with ATP, radiolabeled rhodopsin appears in
vesicle-enriched fractions 4-6 of the gradient. By contrast, ATP
depletion arrests rhodopsin in the heavy fractions (9, 10, 11, 12) enriched in Golgi
and TGN membranes. The identity of these compartments was previously
demonstrated by their galactosyltransferase and sialyltransferase
activity, respectively (Deretic and Papermaster (1991, 1993a); see also Fig. 3). The distribution of newly synthesized rhodopsin in the
gradient fractions after in vitro vesicle formation in the
presence of ATP closely parallels its distribution after in vivo vesicle formation (Fig. 1), suggesting that all the
components necessary for this process are present in the
photoreceptor-enriched PNS. In the presence of ATP, fractions 4-6
contain 32% of newly synthesized rhodopsin compared with 15% in
the absence of ATP. The amount of the post-Golgi vesicles formed in the
absence of ATP represents that which is initially formed in vivo during the 60-min pulse prior to breaking the cells (see Fig. 4).
Figure 3: Membrane proteins of the TGN are retained during cell-free vesicle budding. The distribution of sialyltransferase, a resident membrane protein of the TGN, is not affected by vesicle budding in a cell-free assay at 22 or 0 °C and resembles its distribution after in vivo vesicle formation (control). The bulk of sialyltransferase is associated with fractions 7-11, and very little is found in vesicle-enriched fractions 4-6. After the completion of an in vitro or an in vivo chase, each subcellular fraction is assayed for sialyltransferase activity, and the distribution of the enzyme is expressed as a percentage of total activity recovered in retinal subcellular fractions.
Figure 2: ATP-dependent vesicle formation progresses at low temperature in frog retinal PNS. Top panel, after 60 min of pulse labeling, whole retinas are chased at 22 °C (in vivo, 1) as in Fig. 1, and retinal PNS is chased in the presence (+ATP, 2) or the absence of ATP (-ATP, 3) at 0 °C (in vitro). Autoradiograms of the rhodopsin regions of the SDS-PAGE are shown as in Fig. 1. Bottom panel, densitometric scans of the autoradiograms as in Fig. 1. The different distribution of radiolabeled rhodopsin in the presence of an ATP-depleting system at 0 °C (this figure) versus 22 °C (Fig. 1) has been reproducibly observed in three separate experiments.
Fractions 7-10 are enriched in sialyltransferase activity contributed by the TGN membranes. These are also the fractions that accumulate radiolabeled rhodopsin during the chase on ice in the absence of added ATP (see Fig. 2).
Figure 5:
Post-Golgi vesicles formed in vivo and in vitro have identical content of
membrane-associated small G-proteins. rab11 is also a vesicle protein.
After subcellular fractionation and high speed centrifugation,
membrane-associated GTP-binding proteins from two frog retinas were
detected by two-dimensional gel electrophoresis and
[P]GTP overlays (A-D). Whereas
the Golgi-enriched fraction has a more complex pattern (A)
post-Golgi vesicles formed in vitro at 22 (B) or 0
°C (C) are identical to the vesicles formed in vivo in intact retinal cultures (D). Previously identified
proteins rab6 and rab8 are indicated by open and closed
arrowheads, respectively. E, the post-Golgi vesicles also
contain rab11. rab11 is identified in this panel by the anti rab11
antiserum (Urbéet al., 1993); it is
also indicated with an arrow in D. The subcellular
distribution of rab11 parallels that of rab6 in retinal photoreceptors
(data not shown).
Figure 6:
Two-dimensional gel pattern of post-Golgi
vesicle proteins reveals significant reduction in complexity of these
membranes (B) compared with the Golgi-enriched fraction (A). Silver-stained proteins separated by two-dimensional gel
electrophoresis of vesicle membranes reveal several previously
identified vesicle proteins: the -subunit of transducin (arrow),
A and
B crystallin (open and closed arrowheads, respectively), and membrane-bound actin (asterisk), as well as a number of unknown proteins. The
identity of the indicated proteins has been confirmed by
immunoblotting. The
subunit of transducin, detectable by
immunoblotting, is not easily detectable here, probably due to its low
staining with silver (Hamm and Bownds, 1986). The two proteins at the
basic end of the gel in B, indicated by the notches on the right side, are 97-kDa and 30-kDa molecular mass
standards.
Figure 7:
Post-Golgi vesicles formed in the
cell-free system are morphologically identical to the vesicles formed in vivo and differ from Golgi-enriched fraction. Electron
micrographs of the thin sections through the pellet obtained after in vitro vesicle formation, sedimentation of fraction 5 or 12
membranes, and embedding in Epon are shown. A, a population of
small vesicles is a major component of fraction 5. They are of the same
size (300 nm) and appearance as vesicles formed in vivo,
which we have previously characterized. B, fraction 12 is very
heterogeneous. Occasional profiles that may represent cross-sections of
the Golgi cisternae can be seen (open arrow). In addition this
fraction contains mitochondria, smooth membranes, and multivesicular
bodies. Bar, 0.3 µm.
Rhodopsin-bearing post-Golgi vesicles formed in vivo in retinal cultures contain rhodopsin in the membrane with its carboxyl-terminal domain exposed to the cytoplasm and amino-terminal domain inside the vesicle (Deretic and Papermaster, 1991). We have probed the orientation of rhodopsin in the vesicles that are formed in vitro at 22 or 0 °C using mAb 11D5 to the carboxyl-terminal domain of rhodopsin and found that they contain rhodopsin inserted in the membrane with this domain exposed at the cytoplasmic side and susceptible to limited proteolysis with thermolysin (Fig. 8). This suggests that in vitro formed vesicles are topologicaly identical to the vesicles formed in vivo.
Figure 8: Topological analysis of in vitro formed post-Golgi vesicles probed with thermolysin and an mAb 11D5 to the carboxyl-terminal cytoplasmic domain of rhodopsin. Blots of the untreated and thermolysin-treated vesicles were probed with the mAb 11D5, whose antigenic site is within nine amino acids from the COOH-terminus of rhodopsin (Deretic and Papermaster, 1991). Proteolytic cleavage of the carboxyl-terminal domain of rhodopsin by thermolysin destroys the mAb 11D5 binding site on the vesicles formed in the cell-free assay at 22 or 0 °C.
Figure 9:
Extraction of rab proteins by rab GDI
arrests unprocessed rhodopsin in the Golgi. To remove membrane-bound
rab proteins, radiolabeled retinal PNS was preincubated with 200
µM GDP for 1 h at 22 °C, and then 2 µM GDI was added. the preincubation was continued for 30 min at 30
°C, followed by 2 h of cell-free chase at 22 °C. A,
[P]GTP overlays of the post-Golgi vesicle
membrane proteins separated by two-dimensional gels show >60%
reduction in membrane associated rab6 and rab8 (open and closed arrowheads, respectively). The more basic of the two
ubiquitous unidentified proteins that co-migrates with rab11
(Urbéet al., 1993) remains unchanged (arrow). B, [
P]GTP overlays of
soluble proteins separated by SDS-PAGE show a significant increase of
small G-proteins in the cytosol after GDI extraction. Immunoblotting
with anti-rab6 antibody and quantitative analysis of the blots
developed using the ECL system reveals a 3-fold increase in soluble
rab6. C, removal of rab proteins by GDI inhibits vesicle
formation (fractions 4-6) and arrests slowly migrating rhodopsin
in the Golgi (fractions 11 and 12). Subcellular fractions were pooled
according to the kinetics of their acquisition of newly synthesized
rhodopsin, and membrane proteins were separated by SDS-PAGE.
Autoradiograms of the gels are shown. R, rhodopsin. The arrows indicate rhodopsin dimers and trimers. The gels were
exposed for a short period so that the autoradiograms reveal only
rhodopsin and its multimers as described previously (Deretic and
Papermaster, 1991).
Figure 10:
rab GDI affects rhodopsin transport in a
concentration-dependent manner. Retinal PNS was preincubated with 200
µM GDP for 1 h at 22 °C followed by 30 min at 30
°C with increasing concentrations of GDI. After SDS-PAGE the
distribution of radiolabeled rhodopsin was determined by a
PhosphorImager densitometer. The experiment was repeated three times.
The GDI-induced decrease in radiolabeled rhodopsin content of the
vesicular pool in fractions 4-6 (IC = 0.3
µM GDI) is accompanied by an equivalent increase in the
Golgi-enriched fractions 11 and 12.
Figure 11:
A monoclonal antibody 11D5 to the
carboxyl-terminal cytoplasmic domain of frog rhodopsin inhibits
post-Golgi vesicle formation and arrests rhodopsin in the TGN. A, radiolabeled retinal PNS was preincubated with 100 µg
of mAb 11D5 or control antibody for 30 min at 0 °C before the 2-h
cell-free chase. Subcellular fractions were pooled as described in the
legend to Fig. 9. Autoradiograms of the SDS-PAGE are shown.
Although mAb 11D5 blocks vesicle formation, control antibody has no
effect. R, rhodopsin (35 kDa). The arrow indicates rhodopsin dimer. A 45-kDa radiolabeled protein visible
in this autoradiogram and other less abundant (not shown)
vesicle-associated proteins are also arrested in fractions 7-10
by the action of anti-rhodopsin antibody. B, the distribution
of radiolabeled rhodopsin was measured by the PhosphorImager
densitometer in four separate experiments. The data are presented as
the means ± S.E. In the control (with ATP at 22 °C) in
addition to the vesicles formed during the pulse,
16% of total
radiolabeled rhodopsin accumulates in the vesicle fraction during the
cell-free chase. mAb 11D5 and its Fab fragments diminish this
additional vesicle formation to
2%. Therefore, vesicle formation
during the chase in the presence of mAb 11D5 is inhibited >85%.
Concurrently, fractions 7-10 enriched in the TGN membranes (see Fig. 3) accumulate radiolabeled rhodopsin. Two additional
antibodies to other photoreceptor proteins and the Fab fragments from
the control antibody have no effect on vesicle formation (not
shown).
To test if potential antibody cross-linking changes the sedimentation of the vesicles rather than inhibits their budding, we also tested the mAb 11D5 Fab fragments. 11D5 Fab fragments inhibit vesicle formation in the same fashion as the whole antibody (Fig. 11B), whereas control Fab fragments have no effect (data not shown). Because Fab fragments can not cause membrane cross-linking, this implies that mAb 11D5 inhibits rhodopsin transport. The presence of the antibody in the cell-free assay does not appear to interfere with the intra-Golgi transport because radiolabeled rhodopsin is found in lighter fractions (7, 8, 9, 10) compared with its arrest in fractions 11 and 12 by GDI. We conclude that mAb 11D5 exhibits its inhibitory effect on the vesicle formation from the TGN. This suggests that the intracellular sorting machinery recognizes the cytoplasmic domain of rhodopsin upon its appearance in the TGN, where it is sorted to the appropriate vesicular carriers.
Establishment of a cell-free system that reconstitutes rhodopsin-bearing post-Golgi vesicle formation represents the first step toward understanding the molecular mechanisms of polarized sorting in rod retinal photoreceptor cells. Studies of intracellular trafficking of newly synthesized rhodopsin may also provide a more general insight, because rhodopsin is a member of the large family of seven-helix transmembrane receptors. We find that rhodopsin-bearing post-Golgi vesicles can form in the frog retinal cell-free system under physiological conditions and that vesicle budding is accompanied by the retention of resident membrane proteins within the TGN. Similar conditions for cell-free post-Golgi vesicle formation in other cells have been reported previously (Tooze and Huttner, 1990; Tooze et al., 1990; Salamero et al., 1990; Jones et al., 1993). However, in contrast to the cells or cell-lines that originate from the warm blooded animals, frog-derived tissues also appear to be able to maintain membrane transport at very low temperatures.
Sustained intracellular transport at 0 °C may be due to the
preservation of membrane fluidity at low temperatures in frog
photoreceptors. We have recently found that the polyunsaturated
docosahexaenoic acid, a major contributor to the exceptionally high ROS
membrane fluidity, incorporates into transport membrane phospholipids
at the site of vesicle formation at 22 °C. ()It will be
of interest to determine the temperature dependence of this process to
define the role of lipid remodeling in intracellular transport and the
post-Golgi vesicle budding.
A potential chaperone activity of A
and
B-crystallins may also contribute to the stability of
post-Golgi vesicle formation at low temperature. These heat shock
proteins associate uniquely with rhodopsin-bearing vesicles and may
provide protection against temperature-induced stress (Deretic et
al., 1994).
The presence of excess (2 µM) rab GDI in the retinal cell-free assay probably inhibits intra-Golgi transport by depleting a rab protein pool present on the Golgi membranes and inhibiting recycling and recruitment of soluble rabs. Similar effects of rab GDI on the intra-Golgi transport in other cells have been reported (Elazar et al., 1994; Peter et al., 1994). The pattern and the distribution of newly synthesized rhodopsin upon removal of rab proteins in retinal cells greatly resembles the effect of short (30 min) exposure to brefeldin A which results in the disruption of the Golgi and inhibition of rhodopsin processing (Deretic and Papermaster, 1991). The similarity between GDI- and brefeldin A-induced arrest of transport suggests that removal of rab proteins interferes with the glycoprotein processing in the Golgi (and, by inference, possibly its structure as well), which ultimately leads to reduced vesicle formation. rab6 is extracted from the photoreceptor membranes upon treatment with GDI (Fig. 9). Because rab6, as well as rab1, appears to be critical for the integrity of the Golgi complex (Martinez et al., 1994; Peter et al., 1994), their collective removal from the photoreceptor Golgi by rab GDI may contribute to the arrest of transport there. Additional experiments will be necessary to determine the role of rab6 and other post-Golgi vesicle-associated rab proteins in the budding and targeting of rhodopsin-bearing post-Golgi vesicles.
All rhodopsin-bearing vesicle-associated rab proteins are properly sorted to the post-Golgi vesicles formed in vitro. However, the amount of rab8 detectable by GTP-overlays appears to be diminished compared with the vesicles formed in vivo. rab6 remains unchanged. A possible explanation for this finding is that the cell-free assay reconstitutes vesicle budding but may not reconstitute vesicle targeting and fusion. In this case the difference between the two rab proteins would be consistent with our previous conclusion that rab8 acts at the later step of rhodopsin transport than rab6 (Deretic et al., 1995).
The principal unique finding presented in this study is the important role of rhodopsin in photoreceptor post-Golgi vesicle formation. In the cell-free assay, mAb 11D5 to the COOH-terminal cytoplasmic domain of rhodopsin and its Fab fragments appear to block transport of rhodopsin and its associated post-Golgi vesicle proteins at the exit from the TGN. This finding is consistent with our previous proposal that rhodopsin not only regulates its polarized sorting but also the sorting of ROS proteins transducin and cGMP phosphodiesterase, its partners in signal transduction (Deretic and Papermaster, 1991). Moreover, additional evidence for the important regulatory role of rhodopsin has been provided by Roof et al.(1994), who showed that in transgenic mice mis-sorting of mutant rhodopsin also leads to delocalization of other nonmutant ROS proteins.
Sorting into
specific post-Golgi vesicles in photoreceptors may be accompanied by
the binding of the cytosolic coat proteins that are presumed to be
essential to deform the membrane and drive the budding process. This
coat could be assembled at or near the cytoplasmic surface of
rhodopsin, or perhaps rhodopsin molecules may bind integral TGN
membrane protein(s) responsible for the recruitment of coat proteins
and/or regulation of sorting to the appropriate vesicular carriers. In
either case, antibody bound to the cytoplasmic surface of rhodopsin
could prevent coat binding to its membrane receptor(s). Preliminary
experiments show that protein p200, a candidate for a coat protein that
regulates vesicle budding from the TGN (Narula et al., 1992;
Ladinsky et al., 1994) associates with photoreceptor TGN
membranes but not the post-Golgi vesicles and could participate in
vesicle budding. ()
It is possible that some of the coat proteins are photoreceptor-specific. This is consistent with reports of nonpolarized expression of rhodopsin in polarized kidney-derived cell lines, which suggest that these cells lack appropriate machinery to sort rhodopsin to one domain of the plasma membrane (Oprian et al., 1987; Nathans et al., 1989). Although we find that proteins that regulate transport to the basolateral plasma membrane in epithelial cells, such as rab8, may be involved in rhodopsin transport in rods, (Deretic et al., 1995), it is possible that photoreceptors use additional molecular information to sort rhodopsin.
It is difficult to assess which part of the cytoplasmic domain of rhodopsin is responsible for the sorting function because bound mAb 11D5 or its Fab fragments probably render the entire surface unavailable for interactions with other proteins. However, it is possible that the extreme COOH-terminal that binds the antibody also contains the sorting signal. The five COOH-terminal amino acids QVA(S)PA are highly conserved among different species. This domain of rhodopsin is in close proximity to the domains that participate in light-dependent G-protein activation or subsequent phosphorylation and inactivation by rhodopsin kinase; however; it has not been implicated in any light-dependent function (Weiss et al., 1994; Chen et al., 1995; Sung et al., 1994; Hargrave and McDowell, 1992). Mutations in the COOH-terminal domain severely impair rhodopsin function and lead to rapid retinal degenerations and blindness in patients with autosomal dominant retinitis pigmentosa (Nathans, 1994). Recently, Sung et al.(1994) have shown that transgenic mice expressing mouse rhodopsin that lacks five COOH-terminal amino acids accumulate this but not the endogenous rhodopsin in the lateral photoreceptor plasma membrane, which supports the role of the COOH-terminal in sorting to or retention in the ROS.
Our data indicate that the cytoplasmic domain of rhodopsin interacts
with the intracellular sorting machinery at the exit from the TGN. We
are currently attempting to map the region of rhodopsin responsible for
this interaction using synthetic peptides derived from the cytoplasmic
domain of bovine and frog rhodopsin. ()Our future efforts
will also be focused on identification of the membrane and/or coat
proteins that interact with rhodopsin upon exit from the TGN. These
factors should further define the mechanism of development of
photoreceptor polarity, which may involve the modification of the
sorting machinery common to other polarized cells, or the development
of additional sorting stations to fulfill the maintenance of three
highly polarized domains in this specialized neuron.