(Received for publication, May 11, 1995)
From the
In Caco-2 cells, aminopeptidase N is transported to the apical membrane from the trans Golgi network by both the direct and the indirect pathway (Matter, K., Brauchbar, M., Bucher, K., and Hauri, H.-P.(1990) Cell 60, 429-437). The aim of this study was to determine the importance of the transmembrane or cytoplasmic domain of aminopeptidase N for transport of aminopeptidase N by the indirect pathway by analysis of mutated forms of aminopeptidase N recombinantly expressed in Caco-2 cells. A tail-less and two secretory forms of aminopeptidase N, all deprived of the cytoplasmic tail, were transported to the basolateral plasma membrane in proportions equivalent to the wild type enzyme. This shows that no cytoplasmic basolateral sorting signal is involved in directing aminopeptidase N to the basolateral plasma membrane. Both the wild type and the tail-less aminopeptidase N were transcytosed from the basolateral to the apical plasma membrane, whereas no transcytosis of two secretory forms could be detected, showing that the transmembrane domain is important for efficient transcytosis to take place. A significant difference in transcytosis kinetics of the human and the porcine wild type aminopeptidase N was observed. This indicates that transcytosis of aminopeptidase N from the basolateral to the apical membrane does not occur by default transport but involves an active sorting mechanism.
The plasma membrane of epithelial cells is divided into an
apical and a basolateral domain by tight junctions. Polarized
epithelial cells are able to selectively direct membrane-bound proteins
to either of these membrane domains. It has been shown that there are
at least two pathways to the apical membrane: a direct pathway from the
trans Golgi network and an indirect pathway from the trans Golgi
network to the basolateral membrane followed by transcytosis to the
apical domain (for a review, see Rodriguez-Boulan and Powell(1992)).
Proteins transported to the apical and basolateral plasma membrane in
polarized cells are generally believed to be guided by sorting signals.
The best studied example of a protein using the indirect pathway is the
polymeric immunoglobulin receptor in Madin-Darby canine kidney (MDCK) ()cells. A cytoplasmic basolateral sorting signal directs
the polymeric immunoglobulin receptor to the basolateral domain
(Casanova et al., 1991a; Aroeti et al., 1993). Then,
two cytoplasmic endocytosis signals ensure rapid endocytosis (Okamoto et al., 1992) to endosomes where the receptor may recircle to
the basolateral membrane domain again guided by the basolateral sorting
signal or transcytose to the apical membrane domain (Aroeti and Mostov,
1994). Transcytosis is promoted by ligand binding (Hirt et
al., 1993; Song et al., 1994) and phosphorylation of
Ser-664 (Hirt et al., 1993; Casanova et al., 1990).
The transport to the apical plasma membrane seems to be signal mediated
by ill-defined part(s) of the extracellular domain (Mostov et
al., 1987).
Another protein, dipeptidyl peptidase IV, can also use the indirect pathway to achieve an apical expression in both MDCK cells (Casanova et al., 1991b) and Caco-2 cells (Matter et al., 1990). It has been reported that dipeptidyl peptidase IV in MDCK cells carries a basolateral sorting signal on the cytoplasmic domain and an apical sorting signal on the extracellular domain (Weisz et al., 1992). In both MDCK cells (Weisz et al., 1992) and Caco-2 cells (Matter et al., 1990), only part of the newly synthesized dipeptidyl peptidase IV molecules use the indirect pathway, whereas the rest uses the direct pathway to the apical membrane.
We have previously shown that aminopeptidase N (APN) carries an apical sorting signal on the extracellular domain directing the protein to the apical side in MDCK cells (Vogel et al., 1992a, 1992b), and thus, in this respect, APN resembles both dipeptidyl peptidase IV and the polymeric immunoglobulin receptor. In MDCK cells, it has been shown that the majority of newly synthesized APN molecules are transported via the direct pathway (Wessels et al., 1990). In Caco-2 cells, APN is transported to the apical side via both the direct and the indirect pathways (Matter et al., 1990; Le Bivic et al., 1990). We have set out to investigate the sorting signals directing the indirect transport of APN in Caco-2 cells with special attention to possible sorting signals in the cytoplasmic tail.
In the present study, we found that the transport of APN to the basolateral membrane is independent of sorting signals in the cytoplasmic domain. However, the transmembrane domain is necessary for transcytosis of APN from the basolateral to the apical plasma membrane. In addition, we show that the Caco-2 cell line, which is of human origin, is able to transcytose human APN more efficiently than porcine APN.
The construction of the anchor-minus and the anchor/stalk-minus human APN cDNA was previously described (Vogel et al., 1992a, 1992b). The construction of the cDNA for the tail-less and Tyr-6 porcine APN was introduced by polymerase chain reaction and sequenced to verify the construction. All cDNAs were introduced into the eukaryote expression vector pTEJ-4 (Johansen et al., 1990). Cells were transfected with 20 µg of pTEJ-4 containing the relevant cDNA and 2 µg of pSV2-neo (Southern and Berg, 1982).
Figure 1: The N-terminal sequences of the wild type porcine APN, porcine APN mutated in tyrosine 6 (Tyr-6), porcine APN with deleted tail (tail-less), wild type human APN, anchor-minus human APN, and anchor/stalk-minus human APN. The transmembrane-spanning parts are underlined by a singleline, and the hemagglutinin signal peptide is underlined by a doubleline. The hemagglutinin signal peptide cleavage site is marked by an arrow.
It has previously been shown that in Caco-2 cells about 40% of the endogenous human APN is transported by the indirect pathway (Matter et al., 1990), leaving about 60% to the direct pathway using cell culture conditions (5-13 days post-confluent) similar to ours (3-6 days post-confluent). Using 15-20 days post-confluent cells, 60-70% of the endogenous APN was transported by the indirect pathway (Le Bivic et al., 1990). The present study thus shows that Caco-2 cells transport the secretory forms of APN to the plasma membrane with a distribution between the apical and the basolateral side very similar to what has been reported for the endogenous membrane-bound enzyme. The cells are, however, unable to transcytose the basolateral pool of the secretory forms to the apical side. This is in contrast to what has been reported for the endogenous membrane-bound enzyme (Matter et al., 1990; Le Bivic et al., 1990). This points to an important role for the cytoplasmic and/or the transmembrane domain for efficient transcytosis of APN in Caco-2 cells.
The tail-less, Tyr-6, and wild type porcine APN cDNAs were
transfected into Caco-2 cells, and stable clones were isolated. The
clones were screened using [S]methionine
labeling followed by immunoprecipitation, and clones expressing
tail-less, Tyr-6, and wild type APN were identified. Pulse-chase
experiments showed that the tail-less and the Tyr-6 APN were converted
from the high mannose to the complex glycosylated form with kinetics
very similar to that of the wild type porcine APN (data not shown).
To determine the polarized distribution of the recombinant proteins,
filter-grown transfected Caco-2 cells were incubated at 4 °C with
saturating concentrations of antiserum G43 either on the apical or the
basolateral side, followed by incubation with I-labeled
Fab fragments. For each construct, two clones were investigated with
essentially the same result. Total cellular extracts were counted on a
counter. Quantitation of two to five experiments showed (Table 1) that about 70% of the cell surface expressed wild type,
tail-less, and Tyr-6 porcine APN located on the apical side. These
results show that the cytoplasmic tail has no influence on the steady
state distribution of the porcine APN between the apical and the
basolateral membrane.
This difference between the human and
the porcine APN was further investigated by studying the appearance of
newly synthesized proteins on the two cell surface domains. Cells were
pulse labeled with [S]methionine for 20 min, and
after various chase times the plasma membrane proteins present in
either the apical or basolateral domain were biotinylated. Biotinylated
APN was purified by immunoprecipitation using protein A-agarose,
released from the beads by boiling, and reprecipitated with
streptavidin-agarose. The resulting purified antigens were then
analyzed on SDS-PAGE gels and quantitated. Caco-2 cells expressing
recombinant wild type human APN were investigated, and the enzyme was
found to be transported initially to both the apical and the
basolateral membrane with a clear transient pool at the basolateral
membrane (Fig. 2, panelA). The basolateral
fraction disappeared after 3 h of chase, which resembles what has been
reported for the endogenous human APN (Matter et al., 1990; Le
Bivic et al., 1990). The porcine APN was also transported to
the two membranes simultaneously (Fig. 2, panelB), and a transient pool of APN was observed at the
basolateral side, albeit its disappearance from the basolateral side is
slower and less effective than that of the human APN. The basolateral
pool is still clearly visible after an overnight chase (data not
shown). Caco-2 cells are thus able to polarize the human APN more
efficiently than the porcine APN.
Figure 2:
Appearance at the cell surface of newly
synthesized recombinant wild type human, wild type porcine, or
tail-less APN. Tight monolayers of Caco-2 cells grown on filters were
pulse labeled with [S]methionine for 20 min and
chased for the time indicated. The apical or the basolateral side was
then biotinylated with s-NHS-biotin. APN was immunoprecipitated from
the cell lysates. Biotinylated APN was recovered by adsorption to
streptavidin-agarose and analyzed by SDS-PAGE and quantitated. The
results are expressed as a percentage of the total amount at the time
of maximal expression at the cell surface. At least two experiments
were done. Caco-2 cells transfected with wild type human (A),
wild type porcine (B), and tail-less (C) APN cDNA are
shown (apical (hollowcircle) and basolateral (filledcircle)).
To assess directly whether the newly
synthesized wild type and tail-less porcine APN that appear transiently
on the basolateral membrane are routed to the apical cell surface, we
used the following protocol. Wild type or tail-less porcine APN
expressing cells, grown on filters, were pulse labeled with
[S]methionine for 20 min, chased for 60 min, and
then biotinylated on the basolateral surface with the cleavable reagent
s-NHS-SS-biotin at 37 °C for 20 min followed by a second chase for
0 or 4 h. At the indicated times, the cells were reduced twice with
MESNA from the apical side or from the basolateral side. These
experiments show that both wild type and tail-less APN, biotinylated
from the basolateral side, can be partly reduced from the apical side (Fig. 3) after a 4-h chase. A control experiment showed that
without a second chase, the biotin could be reduced from the
basolateral side but not from the apical side. Thus, the wild type and
tail-less porcine APN molecules are transcytosed from the basolateral
to the apical side to approximately the same degree.
Figure 3: Transcytosis of wild type and tail-less porcine APN in Caco-2 cells. Cells were pulsed for 20 min and chased for 60 min. The cells were thereafter biotinylated from the basolateral side with s-NHS-SS-biotin, a cleavable analogue of biotin, at 37 °C and further chased for 0 or 4 h. After this second chase, cells were reduced with MESNA from the apical (A) or basolateral (B) side or reduction was omitted (none). The biotinylated aminopeptidase N was purified by the double immunoprecipitation and streptavidin precipitation protocol described in the legend to Fig. 2, analyzed by SDS-PAGE gels, and quantitated. The means of at least two independent experiments are shown.
It is well known that both porcine and human APN form homodimers (Danielsen, 1994, Jascur et al., 1991). Thus, it is possible that the recombinant and endogenous APN expressed together in Caco-2 cells in this study form heterodimers. However, the anchor-minus and anchor/stalk-minus APN secreted from Caco-2 cells are unlikely to represent molecules that pass through the cell in a heterodimer state (between soluble and membrane-bound APN) since we have previously experienced, during purification of the enzyme, that the dimer is very stable (Sjöström and Norén, 1982). When analyzing the recombinant-expressed porcine aminopeptidase N, it is possible that maximally of the molecules (estimated from the expression level of aminopeptidase enzymatic activity of non-transfected and transfected cells) are in a heterodimer state and thus, if present, constitute a minor fraction.
It is not known whether the transcytotic transport from the basolateral to the apical domain is a signal-mediated process, but the finding in this study that the porcine and the human APN are transcytosed with different kinetics provides evidence that this is not a default pathway, and hence active sorting is involved. The signal involved is not located in the cytoplasmic part since the human and the porcine APN transcytose with significantly different kinetics despite a 100% identity in the cytoplasmic tail. However, the transmembrane domain is necessary for the ability to transcytose efficiently, as seen by the lack of transcytosis of the soluble forms. Whether a transcytosis signal is located in the transmembrane domain or it is merely the membrane attachment that is important to ensure efficient endocytosis is not yet known.
In this study, we found that the Caco-2 cell line that is
derived from a human colon adenocarcinoma is able to sort human APN
more efficiently than porcine APN. When the porcine APN cDNA was
expressed in MDCK cells, 92 ± 5% of the total surface-expressed
APN was located on the apical domain, ()suggesting that the
difference in polarization in Caco-2 cells is not due to accidentally
introduced mutation(s) in the cloned porcine cDNA. It could be
hypothesized that polarization is an old and well conserved trait, but
the present study indicates that the mechanisms leading to polarization
have undergone some modification during evolution. The differences
between the efficiency in sorting of the human and porcine APN may
provide an opportunity to map the sorting signal operating on the
transcytotic pathway in Caco-2 cells. There is 79% identity between the
human and the porcine APN amino acid sequences (Delmas et al.,
1992). A range of chimeras between the human and porcine APN has
already been analyzed in MDCK cells to map a virus binding side on APN
(Delmas et al., 1994), and they were found to be well
expressed on the cell surface indicating a correct three-dimensional
structure of the chimeric molecules that allows cell surface
expression.