A Different Intracellular Distribution of a Single Reporter
Protein Is Determined at Steady State by KKXX or KDEL
Retrieval Signals*
Lavinia V.
Lotti
,
Giovanna
Mottola§,
Maria R.
Torrisi¶
, and
Stefano
Bonatti§**
From the
Istituto Nazionale Ricerca sul Cancro di
Genova, Sezione di Biotecnologie, Viale Regina Elena 324, 00161, Rome,
the ¶ Dipartimento di Medicina Sperimentale e Patologia,
Università di Roma "La Sapienza," Viale Regina Elena 324,
00161, Rome, the
Istituto Dermatologico San Gallicano, Via San
Gallicano, 00100, Rome, and the § Dipartimento di Biochimica
e Biotecnologie Mediche, Università di Napoli "Federico II,"
via S. Pansini 5, 80131 Naples, Italy
 |
ABSTRACT |
To establish the specific contribution to protein
topology of KKXX and KDEL retrieval motifs, we have
determined by immunogold electron microscopy and cell fractionation the
intracellular distribution at steady state of the transmembrane and
anchorless versions of human CD8 protein, tagged with KKXX
(CD8-E19) and KDEL (CD8-K), respectively, and stably expressed in
epithelial rat cells (Martire, G., Mottola, G., Pascale, M. C.,
Malagolini, N., Turrini, I., Serafini-Cessi, F., Jackson, M. R.,
and Bonatti, S. (1996) J. Biol. Chem. 271, 3541-3547). The CD8-E19 protein is represented by a single form,
initially O-glycosylated: only about half of it is located
in the endoplasmic reticulum, whereas more than 30% of the total is
present in the intermediate compartment and cis-Golgi complex. In the
latter compartments, CD8-E19 colocalizes with
-coat protein (COP)
(COPI component) and shows the higher density of labeling. Conversely,
about 90% of the total CD8-KDEL protein is localized in clusters on
the endoplasmic reticulum, where significant co-localization with
Sec-23p (COPII component) is observed, and unglycosylated and initially
O-glycosylated forms apparently constitute a single pool.
Altogether, these results suggest that KKXX and KDEL
retrieval motifs have different topological effects on theirs own at
steady state: the first results in a specific enrichment in the
intermediate compartment and cis-Golgi complex, and the latter dictates
residency in the endoplasmic reticulum.
 |
INTRODUCTION |
Many resident proteins of the ER bear short carboxyl-terminal
sequences, KKXX in type I membrane proteins and KDEL in
luminal proteins (1, 2). It has been clearly shown that both motifs are
necessary and sufficient to dictate the recycling of reporter proteins
from the Golgi complex to the ER (3, 4). Key events in this process are
the binding of the KKXX signal, exposed in the cytosol, to
members of the COPI1 coatomer
structure (5, 6), and of the KDEL signal, exposed in the lumen, to a
multispan membrane protein named KDELr (7, 8). Morphological evidence
demonstrates that COPI coatomer and the KDELr are localized in the
Golgi complex as well as in the IC (9, 10); thus, recycling to the ER
could occur from both compartments. The precise pathways of
KKXX and KDEL based recyclings have not been unravelled yet,
and it is not known whether they converge at some point.
The residency in the ER of KKXX and KDEL bearing proteins
does not depend only on the recycling pathway. Evidence in favor of a
direct retention, mediated by weak protein-protein interactions in the
lumen of the ER, has been presented (11-14). Therefore, due to the
likely involvement of retention mechanisms in addition to retrieval
mechanisms, proteins naturally resident in the ER are not the ideal
model system to study the contribution to protein topology of the
KKXX- and KDEL-dependent recycling processes.
To overcome this problem, we decided to use a "neutral" reporter,
the human CD8 glycoprotein (15). CD8 is a type I membrane protein,
uniquely O-glycosylated, which is rapidly transported to the
PM in heterologous cells after transfection (16). Anchorless versions
of CD8, comprising only the luminal domain, are efficiently secreted
(17). Thus, retention determinants, thought to reside in the luminal
portion, should not be present in CD8. Two engineered forms of the
reporter were used in our study: (a) the KKXX
form, CD8-E19, formed by the ecto- and transmembrane domains of CD8 joined to the cytosolic tail of adenovirus E19 protein, which has a
carboxyl-terminal KKMP sequence (18), and (b) the KDEL form,
CD8-K, formed by the ectodomain of CD8 elongated with the SEKDEL
sequence (18). A striking difference was found in the fate of the two
recombinant proteins stably expressed in transfected Fisher rat thyroid
cells (19). Pulse-chase experiments and oligosaccharide analysis showed
that CD8-E19 spends its entire life span within the ER and cis-Golgi
complex (where the O-glycosylation process starts), whereas
newly synthesized CD8-K spends long time routing within the ER and
cis-Golgi complex but eventually escapes from this routing, becomes
terminally glycosylated, and is rapidly secreted. Although these
results implied that in FRT cells the KDEL-based retrieval system is
more leaky than the KKXX-based one, immunofluorescence
observations of the steady-state distribution of the two CD8 forms
suggested that CD8-E19 might have a relatively higher concentration in
the Golgi complex and a correspondingly lower concentration in the ER
than its soluble counterpart CD8-K, with important implications
for the retrieval mechanisms operating between ER and cis-Golgi complex.
In the present study, we have undertaken a quantitative analysis of the
intracellular distribution at steady state of the two tagged reporters
by immunoelectron microscopy and cell fractionation. We indeed found
that CD8-E19 protein is much more present in the IC and cis-Golgi
complex respect to CD8-K, which is largely accumulated in the ER. The
possible relevance of these results for the understanding of the
retrieval mechanisms responsible for the localization of endogenous
protein within ER, IC, and cis-Golgi complex is discussed.
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EXPERIMENTAL PROCEDURES |
Antibodies--
The following antibodies were used: mouse mAb
OKT8 (anti-CD8 protein), from Ortho (Raritan, NJ), mouse mAb N1
(anti-CD8 protein) (19), rabbit polyclonal anti-CD8 (kindly provided by
Dr. M. Jackson, R.W. Johnson Pharmaceutical Research Institute, San
Diego, CA), affinity purified rabbit polyclonal anti-Sec23p (kindly
provided by Dr. J. P. Paccaud, University of Geneva, Switzerland),
rabbit polyclonal anti-
-COP (anti-EAGE, kindly provided by Dr. T. Kreis, University of Geneva, Switzerland), mouse mAb anti-KDELr (kindly provided by Dr. B. Tang, National University of Singapore, Singapore), rabbit polyclonal anti-calnexin (kindly provided by Dr. A. Helenius, Swiss Federal Institute of Technology, Zurich, Switzerland), rabbit polyclonal anti-SSra (kindly provided by Dr. G. Migliaccio, Istituto di
Ricerche di Biologia Molecolare "P. Angeletti," Pomezia, Italy), and rabbit polyclonal anti-ERGIC-p58 (kindly provided by Dr. J. Saraste, University of Bergen, Norway).
Cell Culture, Samples Preparation, and Analysis--
Cell
culture, preparation of cell extracts, immunoprecipitation, SDS-PAGE,
and indirect immunofluorescence were performed as detailed previously
(20-22).
Cell Fractionation--
A detailed description of the procedure
will be presented elsewhere.2
All manipulations were done at 4 °C. Briefly, 7-10 × 107 cells were homogenized by 10 passages through a cell
cracker (8.020-mm chamber, 8.008-mm sphere) in 20 mM
Hepes-KOH, pH 7.3, 120 mM sucrose. A postnuclear
supernatant fraction was obtained by centrifugation at 2500 rpm for 5 min in a Eppendorf centrifuge and loaded on top of a discontinuous
sucrose gradient made up in the same buffer (15, 20, 25, 30, 35, 40, 45, and 60%, w/v). The gradient was spun in a SW 50.1 rotor tube for
1 h at 43,000 rpm in a Beckman ultracentrifuge, and fractions were
collected with a peristaltic pump.
Immunoelectron Microscopy--
All cells were fixed with 1%
glutaraldehyde in phosphate-buffered saline for 1 h at 25 °C
and processed for postembedding immunocytochemistry. In the case of HPL
and KDELr immunolabeling, cells were first permeabilized with digitonin
as described (23) and then fixed. The cells were either partially
dehydrated in ethanol and embedded in LR White resin at 50 °C or
dehydrated in a graded ethanol series by progressively lowering the
temperature, embedded in Lowicryl K4 M at
35 °C, and
polymerized by UV irradiation at
35 °C. Ultrathin sections of LR
White- or Lowicryl K4M-embedded cells were collected on nickel grids
and immunolabeled with anti-CD8 followed by protein A-gold (18 nm; 24).
In double labeling experiments, the sections were first incubated with
anti-CD8 antibody followed by 10 nm of protein A-gold and then treated
with the second set of antibodies followed by 18 nm of protein-A gold.
Control experiments were performed by omission of the primary
antibodies. For HPL labeling, thin sections were incubated at 4 °C
overnight with biotinylated-HPL at a concentration of 20 µg/ml. After
extensive washing, thin sections were incubated with goat anti-biotin
colloidal gold (10 nm) for 1 h at room temperature. The
specificity of the staining was assessed in control experiments adding
50 mM N-acetylgalactosamine to the first
incubation. For double labeling experiments, thin sections incubated
with biotinylated-HPL and anti-biotin colloidal gold were successively
immunolabeled with anti-CD8 followed by protein A-colloidal gold (18 nm). All sections were finally stained with uranyl acetate and lead
citrate before examination with EM. Quantitative evaluation of
immunolabeling was performed as described previously (25).
 |
RESULTS |
The Bulk of Intracellular CD8-E19 and CD8-K Proteins Consists of
Immature Forms--
Detailed studies on the biosynthesis and
glycosylation in FRT cells of CD8, CD8-E19, and CD8-K have been
previously reported (19, 22, 26, 27). As shown in Fig.
1, Western blotting analysis indicates
that untagged CD8 accumulates mostly as the broad mature band of 32-34
kDa, in addition to the unglycosylated and initially glycosylated minor
forms of 27 and 29 kDa, respectively (lane 1); CD8-K was
represented by the unglycosylated 20 kDa form (70% of total) and the
initially glycosylated 22-24 kDa forms (lane 2); and
CD8-E19 accumulated mostly as the initially glycosylated 28 kDa form
(lane 3), which has a half-life longer than 2 days (19).
Therefore, at steady state, almost all CD8-E19 and CD8-K proteins
present in the cells consist of immature forms, no more than initially
glycosylated.

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Fig. 1.
Intracellular reporter and marker forms
expressed by the different transfected FRT clones. Aliquots of 80 µg of total protein of cell lysate were analyzed by SDS-PAGE, blotted
on nitrocellulose filters, and stained by immuno-ECL. Lane
1, FRT-U10 (untagged CD8); lane 2, CD8-E19; lane
3, CD8-K; lanes 4 and 5, parental FRT cells.
Lanes 1-3, anti-CD8 mAb N1; lane 4, anti-calnexin polyclonal antibody; lane 5, anti-ERGIC-p58
polyclonal antibody.
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CD8-E19 Co-localizes with ERGIC-p58 Protein at Light Microscopy
Level--
To ascertain the relative distribution of the two tagged
reporters among ER and IC/cis-Golgi complex, we first performed double immunofluorescence microscopy using calnexin and ERGIC-p58 as marker
proteins, respectively (28, 29). As shown in Fig.
2, both CD8-K and CD8-E19 appeared to
overlap significantly with calnexin (compare panels a and
b to panels e and f), whereas only CD8-E19 showed a pronounced co-localization with ERGIC-p58 protein (compare panels c and d to panels g
and h), thus suggesting an enrichment in the IC/cis-Golgi
area. It is noteworthy that the immunofluorescence labeling pattern of
calnexin, ERGIC-p58, and of several other markers for ER and Golgi
complex was undistinguishable among FRT cells and the clones isolated
after transfection (data not shown), thus excluding alteration of the
ER/Golgi area induced by the expression of the various CD8 forms.

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Fig. 2.
Partial co-localization of CD8-E19 and
ERGIC-p58. Double indirect immunofluorescence analysis of CD8-K
and CD8-E19 cells. Panels a-d, CD8-K cells; panels
e-h, CD8-E19 cells. Panels a, c, e, and g,
anti-CD8 mAb; panels b and f, anti-calnexin
polyclonal antibody; panels d and h,
anti-ERGIC-p58 polyclonal antibody.
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Immunoelectron Microscopical Analysis of the Intracellular
Distribution of CD8-E19 and CD8-K Proteins--
Next, we analyzed at
the ultrastructural level the intracellular localization of CD8-E19 and
CD8-K. As shown in Fig. 3, a and b, CD8-E19 is clearly present on the IC, on one side of
the Golgi complex, and, more dispersed, on the ER; very little labeling was detectable on the PM (not shown). Conversely, most wild type CD8 is
on the PM and the entire Golgi complex (Fig. 3e), very little on the IC (not shown), and almost none on the ER (Fig. 3e). Double labeling with HPL demonstrated that CD8-E19 is
present only in the cis-Golgi complex. HPL binds mostly to
GalNAc-bearing glycoproteins, and the addition of GalNAC, the first
step in the O-glycosylation process, takes place in the
cis-Golgi (30-32). As shown in Fig. 3d, HPL labels only one
side of the Golgi complex and overlaps very well with CD8-E19.
Interestingly, some HPL labeling was detectable on the IC (Fig.
3c). Because the same occurred in CD8-K expressing cells
(see Fig. 4) but never in parental FRT and CD8 expressing cells, this observation suggests that labeling in
the IC was due to recycling tagged CD8 forms. Finally, double labeling
experiments with KDELr, used as marker of the IC/cis-Golgi area, fully
confirmed the distribution of CD8 and CD8-E19 described above (data not
shown).

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Fig. 3.
Immunoelectron microscopical analysis of the
intracellular localization of CD8-E19 and untagged CD8 proteins.
Thin sections of LR White-embedded (a-d) or
lowicryl-embedded (e) cells were immunogold-labeled with
anti-CD8 polyclonal antibody and protein A-colloidal gold conjugates.
Double labeling with anti-CD8 and HPL (c and d)
was also performed. In cells expressing CD8-E19, gold particles were
clearly present on the IC, identified as tubular vesicular structure
(a and b). Labeling was also observed on ER, NE,
and the cis-most cisternae of the Golgi complex, whereas very little
labeling was seen on the trans-Golgi (a). Colocalization of
CD8-E19 (large golds) and HPL (small golds) was observed on the IC
(c) and cis-Golgi cisternae (d, arrows). In cells
expressing untagged CD8 (e), the immunogold labeling was
mostly associated with the PM and with the cisternae of the Golgi
complex. ER and NE appeared virtually unlabeled (e).
G, Golgi complex; M, mitocondrion; Nu,
nucleus; Ly, lysosome. Bars: a, 0.5 µm;
b-e, 0.1 µm.
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Fig. 4.
Immunoelectron microscopical analysis of the
intracellular localization of CD8-K protein. Thin sections of LR
White-embedded cells (a and c-f) were
immunogold-labeled with anti-CD8 polyclonal antibody and protein
A-colloidal gold conjugates. Double labeling with anti-CD8 and HPL
(d and f) was also performed. Immunolabeling
appeared dense and clustered over ER (a) and NE (a,
arrows). Parallel morphological analysis on conventional thin
sections of cells expressing CD8-K showed several protrusions extending
from the NE and clusters of vesicles facing the NE (b,
arrows). Immunolabeling with anti-CD8 was also present on IC
(c) and on cis-Golgi complex (e). PM was
virtually unlabeled (a). Colocalization of CD8-K (large
golds) and HPL (small golds) was observed on IC (d, arrow)
and over cis-Golgi cisternae (f, arrows). Symbols are as in
Fig. 3. Bars: a and b, 0.5 µm; c-f,
0.1 µm.
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A strikingly different distribution was instead observed for CD8-K
protein. It was present in high amounts on the ER and NE and appeared
frequently clustered (Fig. 4a). Careful analysis of the
immunolabeled regions of the ER and NE (see also Fig. 7, c,
e-g) and parallel morphological observations of conventional thin
sections (Figs. 4b and 7d) revealed the presence
of several protrusions extending from the NE and of numerous labeled
vesicles facing the NE. CD8-K was also clearly present in the cis-Golgi and IC (Fig. 4, c and e), as shown by the
co-labeling with HPL (Fig. 4, d and f) and KDELr
(not shown). Conversely, CD8-K was almost absent from the PM and barely
detectable on the trans-side of the Golgi complex (Fig. 4, a
and e). The untagged version of CD8-K, CD8-S, did not
accumulate in the cell (19) and gave only low labeling of the Golgi
complex (data not shown).
To better evaluate the distribution inside the cells of the different
reporters, a quantitation of the immunogold labeling was performed. As
shown in Table I, the absolute labeling
density and its ratio respect to the ER within each cell clone, clearly indicates the enrichment of untagged CD8 in the Golgi complex and of
CD8-K in the ER, whereas CD8-E19 is intermediate between the two
reporters, with a relative higher density in the IC and cis-Golgi
complex. In the absence of stereological measures of the organelles in
FRT cells, we attempted to address the question of the quantitative
distribution of the different reporters by a statistical approach,
assuming that the compartments should be detectable proportionally to
their abundance and size within the cell. Table
II reports the result of this analysis:
almost 75% of untagged CD8 is on the PM, with negligible amount on ER and IC; about 90% of CD8-K is in the ER and nuclear envelope; but only
50% of CD8-E19 is in the ER, because more than 30% is in the IC and
cis-Golgi. Particularly striking appeared the amount of CD8-E19 located
in the IC (about 17%), considering that this compartment is most
likely the smallest of the three.
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Table II
Relative distribution of immunogold labeling
Seven low magnification micrographs of large sections of different
cells from each clone, all cross-sectioning the nucleus, showing large
portion of PM, and coming from a single immunolabeling experiment, were
randomly selected to determine the relative amount of immunolabeling in
the indicated compartments. The measured area of ER + NE + IC + Golgi complex ranged from 6.7 to 7.7 µm for the
three clones. Total number of gold particles counted: CD8, 3049;
CD8-E19, 333; CD8-K, 2341. ND, not
determined.
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Cell Fractionation Analysis of the Intracellular Distribution of
CD8-E19 and CD8-K Proteins--
To evaluate with a different method
the intracellular distribution of the two tagged reporters, we moved to
an analytical cell fractionation approach. Postnuclear supernatant
fractions from CD8-E19- and CD8-K-expressing cells were analyzed on a
discontinuous sucrose gradient, and the distribution of the recombinant
forms of the reporter in the separate fractions was assessed by
SDS-PAGE followed by Western blotting, ECL detection and densitometric quantitation. As shown in Fig. 5, CD8-E19
was present in fractions enriched for ER derived elements as well as
for Golgi stacks membranes, but most striking was the coincidence with
the sedimentation of IC elements, as indicated by the sharp peak of
ERGIC-p58. Conversely, CD8-K was enriched in the ER zone and less
present in the Golgi/IC region of the gradient. Therefore, the results
obtained by cell fractionation are consistent with the evidence
obtained with immunofluorescence and immunoelectron microscopy,
i.e. that CD8-K is more present in the ER and CD8-E19
significantly enriched in the IC.

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Fig. 5.
Cell fractionation analysis of the
intracellular distribution of CD8-K and CD8-E19 proteins.
Postnuclear supernatant fractions were resolved on a discontinuous
sucrose gradient (see under "Experimental Procedures" for details),
and the collected fractions were analyzed by SDS-PAGE followed by
Western blotting developed with antibodies against the indicated
proteins. The amount of each protein per fraction is reported as a
percentage of the total on the gradient. CLNX, calnexin
protein.
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To investigate whether the CD8-K protein molecules residing in the ER
contain Golgi modifications, we compared the distribution on the
gradient of the unglycosylated and initially glycosylated forms. A
version of this experiment in which the CD8-K forms were immunoprecipitated and evidentiated by Coomassie Blue staining of the
SDS-PAGE gel is shown in Fig. 6.
Remarkably, there was no significant difference in the distribution of
the unglycosylated form of CD8-K respect to its initially glycosylated
forms, as expected for a protein quickly recycling from the Golgi
complex.

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Fig. 6.
Cell fractionation analysis of the
intracellular distribution of total (CD8-Kt)
unglycosylated (CD8-Ku), and initially
glycosylated (CD8-Ki) forms of CD8-K
protein. Samples were treated as indicated in Fig. 5, except that
the immunoprecipitated material from each fraction was analyzed by
SDS-PAGE followed by fixation and Coomassie Brilliant Blue staining.
The amount of each protein per fraction is reported as a percentage of
the total on the gradient.
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Intracellular Distribution of COPI and COPII Coatomers--
Strong
evidence suggest that COPI and COPII coated vesicles fulfill different
functions inside the cell. COPI is involved in the retrograde traffic
from the Golgi to the ER, and perhaps also in anterograde intra-Golgi
traffic (5, 6, 33, 34). Although previous data had indicated a role for
COPI in the anterograde traffic from the ER to the Golgi complex (35,
36), it now appears that only COPII is involved in the anterograde
export of vesicles from the ER toward the IC and the Golgi complex
(37-39). As shown in Fig. 7, significant
co-labeling with
-COP (chosen as marker for COPI structure) was
observed in immunoelectron microscopy for both CD8-E19 and CD8-K on the
cis-Golgi and the IC (Fig. 7, a and b);
conversely, no labeling for
-COP was observed on the ER (as well as
on the PM). On the other hand, Sec-23p (representative of COPII
structure) was detected only on the ER of both CD8-E19 and CD8-K
expressing cells in single-labeling experiments; in several cases,
Sec-23p was found on protrusions extending from the ER (Fig.
7g). Given that CD8-E19 shows a low labeling density on the
ER (Table I), we investigated only the possible co-localization of
CD8-K with Sec-23p. Convincing co-labeling of the two proteins was
observed in the NE and the ER (Fig. 7c). Quantitative
analysis of the electron micrographs showed that Sec-23p was almost 6 times enriched in the ER regions containing clusters of CD8-K labeling compared with the regions devoid of CD8-K clusters (not shown). Moreover, several vesicles facing the NE (Fig. 7c, arrow),
and protrusions extending from either the NE or the ER (Fig. 7,
c, e, and f), were clearly co-labeled for Sec-23p
and CD8-K, thus suggesting active export of CD8-K from the ER and a
possible role of COPII in this export. Conventional electron microscopy
clearly indicated the presence in these cells of a non-clathrin coat on many protrusions extending from the ER and NE (Fig. 7d,
arrow) and on adjacent small vesicles (d, arrowheads)
(40).

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Fig. 7.
Colocalization of CD8-E19 protein with
-COP and of CD8-K protein with
-COP and Sec-23p. Thin sections of LR
White-embedded cells expressing CD8-E19 (a) and CD8-K
(b, c, e-g) were double labeled with anti-CD8 and anti-
-COP (a and b) or with anti-CD8 and
anti-Sec-23p (c, e, and f) antibodies.
Colocalization of CD8-E19 (small golds) and -COP (large golds) was
observed on the IC (a) and cis-Golgi cisternae (a,
arrows). Colocalization of CD8-K (small golds) and -COP (large
golds) was also found on the IC and cis-Golgi cisternae (b,
arrows). The ER appears labeled only for CD8-K protein
(b). Colocalization of CD8-K (small golds) and Sec-23p
(large golds) was observed on the NE (c and e)
and ER (f), where the immunolabeling for CD8-K protein is
clustered. Colocalization was also seen over vesicles facing the NE
(e, arrow) and on protrusions extending from the NE
(c, arrows) and the ER (f, arrow). Single
immunolabeling performed with anti-Sec-23p antibody showed gold
particles localized on a protrusion extending from ER (g,
arrow). Parallel morphological analysis on conventional thin
sections of cells expressing CD8-K revealed the presence of a
non-clathrin coat on protrusions extending from the NE (d,
arrow) and on the adjacent vesicles (d, arrowheads).
Symbols are as in Figs. 1 and 2. Bars, 0.1 µm.
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DISCUSSION |
In this study, we examined the role of KKXX and KDEL
retrieval motifs in the intracellular distribution of a reporter
protein at steady state. Previous work, based on immunofluorescence and biochemical analysis after transient transfections (18), could not
address this question. Human CD8 glycoprotein, expressed in FRT cells,
has been chosen as reporter because (a) it does not bear any
known retention/retrieval signal, (b) it readily travels through the secretory pathway, and (c) much information is
available on its posttranslational maturation, as well as on the
glycosylating apparatus of the host cell line. To reach quantitative
conclusion, immunogold electron microscopy was used to extend the
immunofluorescence analysis, and the results were confirmed by cell
fractionation. The evidence obtained demonstrates that the
KKXX and KDEL motifs determine a remarkably different
intracellular localization of the two reporter forms at steady state:
CD8-E19 is significantly enriched in in the IC and cis-Golgi, whereas
CD8-K is almost entirely located in the ER.
Retrieval Motifs and the Early Region of the Secretory
Pathway--
The data indicate that the reporter tagged with the
KKXX motif is located along the entire early segment of the
secretory pathway, i.e. ER, IC, and cis-Golgi complex, more
than confined in the ER. This finding suggests that the steady state
localization of a KKXX-bearing protein within the segment
depends on other factors, such as the presence in the same protein of
export/retention motifs operating in any one of the three compartments.
Evidence in favor of this hypothesis is available, although much of it is only indirect, not based on quantitative measure of intracellular distribution. Indeed, Emp47, a yeast protein that recycles to the ER,
has been found located in the Golgi complex at steady state, and the
deletion mutant lacking the double lysine motif is transported
downstream to the vacuole (41). ERGIC-p53/p58 protein is located in the
ER and IC/cis-Golgi region (29, 42-44); recent work has evidentiated
the crucial role of two phenylalanine residues located at the carboxyl
terminus, essential for exit from the ER, and the presence of an ER
retention signal in the luminal and transmembrane domain of the protein
(45). A similar situation was found in the emp24 protein family
members, present among cis-Golgi and ER, which bear in the cytosolic
tail a double phenylalanine motif and a sequence homologous to an
endocytosis signal (46-48). Moreover, UDP-glucuronosyltransferase
maintains its ER localization when deleted of the double lysine motif,
thus suggesting the presence of a strong ER retention signal (4).
Conversely, for KDEL-bearing proteins, a different situation may exist,
although also in this case much of the evidence is indirect, not based
on quantitative measure of intracellular localization. On one hand, the
endogenous proteins bearing this signal have been all located in the
lumen of the ER, with the exception of exocrine pancreas, where some
secretion has been detected (49). On the other hand, deletion of the
signal resulted in slow secretion, again suggesting the involvement of
retention in the natural localization (11, 12, 50).
Differences between the KDEL and KKXX Determined
Retrieval--
Our results and the previous evidence discussed above
strongly suggest that luminal proteins carrying a KDEL motif are more efficiently located in the ER respect to membrane proteins equipped with a KKXX signal. This behavior could be due to
differences either in the retrieval mechanisms or in the trafficking of
luminal respect to transmembrane proteins. In the first case, the KDEL based retrieval could be more effective because operating more efficiently from the IC. That the IC may represent the first site of
sorting/retrieval after the ER has been claimed (1, 51), but the
absence of specific enzymatic modifications that could mark protein
transiting through this compartment has impaired to reach a firm
conclusion. However, indirect biochemical data may support this
hypothesis. The half-time of glycosylation of CD8-K is about 12 h
(19), and 70% of the intracellular CD8-K is represented by the
unglycosylated form; thus, it is conceivable that the protein exits
from and returns to the ER many times, without reaching the cis-Golgi
complex. Conversely, the findings that CD8-E19 is (a)
localized among ER, IC, and cis-Golgi, but more evenly distributed
among the three compartments, (b) represented by almost a
single, initially glycosylated form, and (c) a long lasting
protein (19) strongly suggest in this case a recycling mechanism that
relies mostly on the Golgi complex. On the other hand, this view does
not deny that CD8-K may be quickly recycled from the cis-Golgi, as
indicated by the finding that at steady state the initially
glycosylated forms of CD8-K do not constitute a separate pool respect
the total (and reinforced by the observation that cell fractionation
analysis after glucosamine pulse-labeling of CD8-K expressing cells
shows the same result; data not shown). In conclusion, a retrieval
mechanism from the IC to the ER that would operate better (or
exclusively) for the KDEL bearing proteins than for the KKXX
ones could well explain the greater accumulation of CD8-K in the ER and
of CD8-E19 in the IC and cis-Golgi complex.
The second hypothesis, that no significant differences exist in the two
retrieval mechanisms because the critical factor is being a soluble or
a transmembrane protein, has to be considered (52). Indeed, soluble
proteins may be at a disadvantage for geometric reasons in a transport
process based on vesicular trafficking. This could in principle explain
the accumulation of CD8-K in the ER, given that the interaction with
the membrane-bound KDELr in the IC and cis-Golgi complex could make the
packaging of the protein in vesicles a different process in the ER
respect to the other two compartments. We consider this hypothesis
unlikely for several reasons. Strong evidence indicates that ER export
does not occur by default (53, 54), but instead it involves sorting and
concentration of the cargo, processes that must depend upon the
interaction of the cargo with receptor/adaptor proteins. The clustering
of CD8-K in the ER and its co-labeling with COPII suggest active export
of the protein. Along these lines, preliminary cell fractionation results of pulse-chase experiments show a rapid movement of newly synthesized CD8-K from the ER to lighter membrane
fractions.3
COPs and ER/IC/cis-Golgi Complex Traffic--
Finally, we looked
at the distribution of COPI and COPII in the cells expressing the
engineered reporters. A complementary situation was found: the first
was detected exclusively in the IC and cis-Golgi complex, the second
only in the ER, often in protrusions and small vesicles very close to
them. Significant co-labeling was observed for both COPs and the two
CD8 forms. Altogether, this analysis fully supports the current view
that COPI is primarily involved in the retrograde traffic to the ER, and possibly in the transport from the IC to the Golgi and within the
Golgi complex, whereas COPII plays an important role only in the
anterograde transport from the ER to the IC.
Conclusion--
The heterologous system we are using to study
in vivo the retrieval processes operating among ER, IC, and
cis-Golgi complex is giving significant information. Our current focus
is on the IC, to address the question of the role of this compartment
in the retrieval process.
 |
ACKNOWLEDGEMENTS |
We thank Drs. A. Helenius, G. Migliaccio, J. Saraste, M. Jackson, L. Orci, and B. L. Tang for the generous gift of
antibody; Drs. N. Borgese and F. Serafini-Cessi for a critical reading
of the manuscript; and Lucia Cutini, Giuseppe Lucania, and Bruno Mugnoz
for excellent technical assistance.
 |
FOOTNOTES |
*
This work was supported in part by grants from Ministero
dell' Universitá e della Ricerca Scientifica e Tecnologica
(MURST) and from Consiglio Nazionale delle Ricerche (Target Project on Biotechnology) (to M. R. T. and S. B.), from Associazione Italiana per la Ricerca sul Cancro (to M. R. T.), and from the Human Capital and Mobility program of the European Community (to S. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed. Tel.: 39-081-7463200;
Fax: 39-081-7463150; E-mail: bonatti{at}unina.it.
2
M. C. Erra, L. Iodice, L. V. Lotti,
M. R. Torrisi, and S. Bonatti, submitted for publication.
3
G. Mottola and S. Bonatti, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
COP, coat protein;
HPL, helix pomatia lectin;
KDELr, KDEL receptor;
IC, intermediate
compartment;
NE, nuclear envelope;
PM, plasma membrane;
ER, endoplasmic
reticulum;
mAb, monoclonal antibody;
PAGE, polyacrylamide gel
electrophoresis.
 |
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