We have previously shown that the ecto-nucleotide pyrophosphatase/phosphodiesterase NPP1 has a restricted basolateral localization in rat hepatocytes, whereas NPP3, another member of the family is apically located (Scott et al., 1997
). The same polarized distributions were observed in MDCK cells transfected with mouse NPP1 or rat NPP3. Therefore, transfected MDCK cells are suitable models in order to study the mechanisms for targeting NPPases to their respective plasma membrane locations in epithelial cells.
The mechanisms for sorting and targeting proteins to the apical and basolateral surfaces are still only partially understood. Sorting of apical proteins may depend on specific interactions of the transmembrane domain or the glycosyl-phosphatidylinositol anchor with membrane microdomains (Simons and Ikonen, 1997
) or on the presence of N- and O-glycans chains within the extracellular domain (Fiedler and Simons, 1995
; Yeaman et al., 1996
; Gut et al., 1998
). However, the sorting signals of NPP3 are still not known. Recent investigations showed that neither glycosylation nor raft association seems to be the targeting mechanism (Rajho Meerson et al., 2000
Sorting of basolateral proteins has been shown to depend on short amino acid sequences located in the cytoplasmic tail (Aroeti et al., 1998
). Different classes of signals have been identified. The most frequent and best-characterized sequences depend on a critical tyrosine residue within a consensus sequence Tyr-X-X-Φ, where Φ is a bulky hydrophobic residue. Most of these tyrosine based-signals may also function as signals for rapid endocytosis. Less common signals include Leu-Leu and di-hydrophobic motifs or short sequences with no apparent consensus.
In this work, we were able to identify the basolateral targeting signal of NPP1 as a short cytoplasmic sequence comprised within the amino acid stretch AAASLLAP, which includes a conserved di-leucine motif. Mutations of either or both leucines largely redirected the protein to the apical surface. Furthermore, addition of the conserved sequence AAASLLAP to the apical protein NPP3 was sufficient to address the protein to the basolateral surface. This result indicates that the basolateral signal of NPP1 is dominant over the apical determinant of NPP3, a feature that has already been recognized for tyrosine-based basolateral signals and basolateral signals with no consensus sequence. In general, fusion of the cytoplasmic tails of basolateral proteins to the transmembrane and ecto-domains of apical proteins or introduction of a basolateral signal resulted in basolateral transport (Brewer and Roth, 1991
; Casanova et al., 1991
; Matter et al., 1992
; Prill et al., 1993
; Thomas et al., 1993
; Kundu and Nayak, 1994
; Thomas and Roth, 1994
; Monlauzeur et al., 1995
; Lin et al., 1997
; Renold et al., 2000
Di-leucine motifs have been generally shown to target proteins to the endosomal/lysosomal system (Sandoval et al., 1994
). Only for the FcRII-2B receptor was a di-leucine motif involved in basolateral sorting (Hunziker and Fumey, 1994
). The di-leucine of the FcRII-2B receptor also mediates entry into the endocytotic pathway, but in the case of NPP1, the di-leucine motif only mediates basolateral sorting because the protein is not rapidly endocytosed. A di-hydrophobic motif Leu-Val in the plasma membrane adhesion protein CD44 (Sheikh and Isacke, 1996
) and a di-leucine motif in the Lutherian glycoprotein (El Nemer et al., 1999
) also appear to function solely as basolateral signals. However, because both these cases are cell-matrix adhesion molecules, they may be stabilized at the plasma membrane and thus be prevented from being internalized. The example of NPP1 suggests that some di-leucine motifs can function as basolateral signals only, whereas others are also able to target proteins to endosomes or lysosomes. Therefore, di-leucine-based signals would form a degenerate family of signals that function in targeting proteins to various intracellular compartments and to the basolateral domain of the plasma membrane, as do tyrosine-based signals (Marks et al., 1997
Because very few basolateral signals of the di-leucine type have been identified, the requirement for basolateral sorting versus endosomal targeting has not been much studied. An unexpected finding was that the deleted mutant NPP1/MLL beginning at the sequence MLL was mainly expressed intracellularly, suggesting that the basolateral signal was converted to an endocytosis signal. Indeed, endocytosis experiments demonstrated that the deleted mutant was internalized, whereas wild-type NPP1 was not, at least during the time of the assay. The fact that in this case very little NPP1 was detectable at the basolateral surface, and yet endocytosis of bound antibody was rapid, suggests that the transit time at the basolateral membrane for this construct is short. However, we cannot exclude that some NPP1/MLL molecules were directly targeted from the trans-Golgi network to the intracellular compartment. Because internalization mainly occurred from the basolateral surface, the new motif probably functions both as a basolateral and endocytotic signal. However, contrary to the AAASLLAP motif, the MLL motif is not dominant over the apical determinants of NPP3, because NPP3/LLAP was mainly expressed at the apical surface, with only little basolateral and intracellular localization.
Analysis of environmental requirements for the di-leucine signal indicated that the amino acids AAAS preceding the two leucines are needed for specific basolateral targeting. Thus, NPP3/AAASLLAP was basolaterally expressed, whereas NPP1/Δ25-30 was mainly intracellular. However, point mutations did not allow us to identify crucial amino acids. Mutation of the central alanine did not affect basolateral sorting, and mutation of the serine into alanine or aspartic acid only caused minimal intracellular localization. Analysis of the amino acid sequences flanking known basolateral di-leucine motifs did not reveal any homology with the AAASLLAP sequence. Therefore, the basolateral signal of NPP1 appears to be a unique motif for specific basolateral sorting.
Many di-leucine–based signals working in endocytosis require charged upstream amino acids to be functional (Pond et al., 1995
; Simmen et al., 1999
; Sandoval et al., 2000
; Shewan et al., 2000
). This does not appear to be the case for the engineered endocytotic signals of NPP1/MLL and NPP1/Δ25-30 in which it seems to be the absence of the AAAS sequence that allows the constructs to be internalized. The sequence AAAS may act either as a retention signal at the plasma membrane or prevent entry of the protein into the endocytotic pathway.
The functional diversity of leucine-based motifs suggests that a family of receptors is able to recognize and discriminate between resembling signals. Furthermore, the similarity between basolateral and endocytotic signals suggests that these receptors may be clathrin adaptor protein (AP) complexes. Four different AP complexes have been identified (Kirchhausen, 1999
). AP-1 and AP-2 complexes bind to the cytoplasmic tail of proteins and subsequently recruit clathrin, at the Golgi apparatus and plasma membrane, respectively. AP complexes have been shown to bind both to tyrosine and di-leucine–based motifs. Binding of tyrosine-based motifs to the μ chains of AP complexes is well established and characterized (Bonifacino and Dell'Angelica, 1999
), but there is some controversy as to whether di-leucine motifs interact with the μ or β chains of AP complexes (Ohno et al., 1995
; Heilker et al., 1996
; Bremnes et al., 1998
; Rapoport et al., 1998
; Rodionov and Bakke, 1998
; Hofmann et al., 1999
Involvement of the AP-1 complex in basolateral sorting of the pIg receptor is supported by coimmunoprecipitation experiments (Orzech et al., 1999
). Furthermore, a new μ subunit of the AP-1 complex, termed μ1B, has been identified as a specific subunit expressed by epithelial cells (Ohno et al., 1999
) and involved in basolateral sorting (Folsch et al., 1999
). However, it is not clear whether the AP-1 complex is able to bind all types of basolateral signals and if it is the only mechanism for basolateral sorting. For instance, the FcRII-2B receptor, whose basolateral signal is a di-leucine, is correctly targeted to the basolateral surface of LLC-PK1 cells (Roush et al., 1998
), although these cells do not express μ1B (Folsch et al., 1999
) and missort many basolateral proteins to the apical surface. The recent discovery of a new family of monomeric adaptors with homology to the γ subunit of the AP-1 complex (Dell'Angelica et al., 2000
; Hirst et al., 2000
) shows that several mechanisms are involved in the sorting of proteins at the trans-Golgi network. Further work will be needed to precise whether interaction with a specific subunit of the AP-1 complex or another mechanism is involved in the sorting of NPP1 and other basolateral proteins with di-leucine motifs and how the flanking amino acids may change the relative affinity of the signal to different binding partners.