Dileucine-based Signals Research Articles

The Double-Leucine Motifs Affect Internalization, Stability, and Function of Organic Anion Transporting Polypeptide 1B1.

Organic anion transporting polypeptide 1B1 (OATP1B1) is specifically expressed at the basolateral membrane of human hepatocytes and plays important roles in the uptake of various endogenous and exogenous compounds including many drugs. The proper functioning of OATP1B1, hence, is essential for the bioavailability of various therapeutic agents and needs to be tightly regulated. Dileucine-based signals are involved in lysosomal targeting, internalization, and trans-Golgi network to endosome transporting of membrane proteins. In the current study, we analyzed the 3 intracellular and 13 transmembrane dileucine motifs (DLMs) within the sequence of OATP1B1. It was found that the simultaneous replacement of I332 and L333 with alanine resulted in a significantly reduced level of the mature form of OATP1B1. The cell surface expression of I332A/L333A could be partially rescued by MG132, as well as agents that prevent clathrin-dependent protein internalization, suggesting that this dileucine motif may be involved in the endocytosis of OATP1B1. On the other hand, I376/L377 and I642/L643, which are localized at transmembrane helices (TM) 8 and 12, respectively, are involved in the interaction of the transporter with its substrates. I642A/L643A exhibited a significantly decreased protein level compared to that of the wild-type, implying that the motif is important for maintaining the stability of OATP1B1 as well.

AP-3–dependent targeting of flippase ATP8A1 to lamellar bodies suppresses activation of YAP in alveolar epithelial type 2 cells

Lamellar bodies (LBs) are lysosome-related organelles (LROs) of surfactant-producing alveolar type 2 (AT2) cells of the distal lung epithelium. Trafficking pathways to LBs have been understudied but are likely critical to AT2 cell homeostasis given associations between genetic defects of endosome to LRO trafficking and pulmonary fibrosis in Hermansky Pudlak syndrome (HPS). Our prior studies uncovered a role for AP-3, defective in HPS type 2, in trafficking Peroxiredoxin-6 to LBs. We now show that the P4-type ATPase ATP8A1 is sorted by AP-3 from early endosomes to LBs through recognition of a C-terminal dileucine-based signal. Disruption of the AP-3/ATP8A1 interaction causes ATP8A1 accumulation in early sorting and/or recycling endosomes, enhancing phosphatidylserine exposure on the cytosolic leaflet. This in turn promotes activation of Yes-activating protein, a transcriptional coactivator, augmenting cell migration and AT2 cell numbers. Together, these studies illuminate a mechanism whereby loss of AP-3-mediated trafficking contributes to a toxic gain-of-function that results in enhanced and sustained activation of a repair pathway associated with pulmonary fibrosis.

Polarized sorting of the copper transporter ATP7B in neurons mediated by recognition of a dileucine signal by AP-1.

Neurons are highly polarized cells having distinct somatodendritic and axonal domains. Here we report that polarized sorting of the Cu(2+) transporter ATP7B and the vesicle-SNARE VAMP4 to the somatodendritic domain of rat hippocampal neurons is mediated by recognition of dileucine-based signals in the cytosolic domains of the proteins by the σ1 subunit of the clathrin adaptor AP-1. Under basal Cu(2+) conditions, ATP7B was localized to the trans-Golgi network (TGN) and the plasma membrane of the soma and dendrites but not the axon. Mutation of a dileucine-based signal in ATP7B or overexpression of a dominant-negative σ1 mutant resulted in nonpolarized distribution of ATP7B between the somatodendritic and axonal domains. Furthermore, addition of high Cu(2+) concentrations, previously shown to reduce ATP7B incorporation into AP-1-containing clathrin-coated vesicles, caused loss of TGN localization and somatodendritic polarity of ATP7B. These findings support the notion of AP-1 as an effector of polarized sorting in neurons and suggest that altered polarity of ATP7B in polarized cell types might contribute to abnormal copper metabolism in the MEDNIK syndrome, a neurocutaneous disorder caused by mutations in the σ1A subunit isoform of AP-1.

Analysis of the Biogenesis of Heparan Sulfate Acetyl-CoA:α-Glucosaminide N-Acetyltransferase Provides Insights into the Mechanism Underlying Its Complete Deficiency in Mucopolysaccharidosis IIIC

Heparan sulfate acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT) catalyzes the transmembrane acetylation of heparan sulfate in lysosomes required for its further catabolism. Inherited deficiency of HGSNAT in humans results in lysosomal storage of heparan sulfate and causes the severe neurodegenerative disease, mucopolysaccharidosis IIIC (MPS IIIC). Previously we have cloned the HGSNAT gene, identified molecular defects in MPS IIIC patients, and found that all missense mutations prevented normal folding and trafficking of the enzyme. Therefore characterization of HGSNAT biogenesis and intracellular trafficking became of central importance for understanding the molecular mechanism underlying the disease and developing future therapies. In the current study we show that HGSNAT is synthesized as a catalytically inactive 77-kDa precursor that is transported to the lysosomes via an adaptor protein-mediated pathway that involves conserved tyrosine- and dileucine-based lysosomal targeting signals in its C-terminal cytoplasmic domain with a contribution from a dileucine-based signal in the N-terminal cytoplasmic loop. In the lysosome, the precursor is cleaved into a 29-kDa N-terminal α-chain and a 48-kDa C-terminal β-chain, and assembled into active ∼440-kDa oligomers. The subunits are held together by disulfide bonds between at least two cysteine residues (Cys(123) and Cys(434)) in the lysosomal luminal loops of the enzyme. We speculate that proteolytic cleavage allows the nucleophile residue, His(269), in the active site to access the substrate acetyl-CoA in the cytoplasm, for further transfer of the acetyl group to the terminal glucosamine on heparan sulfate. Altogether our results identify intralysosomal oligomerization and proteolytic cleavage as two steps crucial for functional activation of HGSNAT.

Optimal Replication of Human Cytomegalovirus Correlates with Endocytosis of Glycoprotein gpUL132

Envelopment of a herpesvirus particle is a complex process of which much is still to be learned. We previously identified the glycoprotein gpUL132 of human cytomegalovirus (HCMV) as an envelope component of the virion. In its carboxy-terminal portion, gpUL132 contains at least four motifs for sorting of transmembrane proteins to endosomes; among them are one dileucine-based signal and three tyrosine-based signals of the YXXØ and NPXY (where X stands for any amino acid, and Ø stands for any bulky hydrophobic amino acid) types. To investigate the role of each of these trafficking signals in intracellular localization and viral replication, we constructed a panel of expression plasmids and recombinant viruses in which the signals were rendered nonfunctional by mutagenesis. In transfected cells wild-type gpUL132 was mainly associated with the trans-Golgi network. Consecutive mutation of the trafficking signals resulted in increasing fractions of the protein localized at the cell surface, with gpUL132 mutated in all four trafficking motifs predominantly associated with the plasma membrane. Concomitant with increased surface expression, endocytosis of mutant gpUL132 was reduced, with a gpUL132 expressing all four motifs in mutated form being almost completely impaired in endocytosis. The replication of recombinant viruses harboring mutations in single trafficking motifs was comparable to replication of wild-type virus. In contrast, viruses containing mutations in three or four of the trafficking signals showed pronounced deficits in replication with a reduction of approximately 100-fold. Moreover, recombinant viruses expressing gpUL132 with three or four trafficking motifs mutated failed to incorporate the mutant protein into the virus particle. These results demonstrate a role of endocytosis of an HCMV envelope glycoprotein for incorporation into the virion and optimal virus replication.

Characterization of basolateral-targeting signals in the neonatal Fc receptor

The neonatal Fc receptor, FcRn, transports proteins through cells, avoiding degradative compartments. FcRn is used in many physiological processes where proteins must remain intact while they move through cells. These contexts include the transport of IgG antibodies from mother to offspring, and the protection of IgG and albumin from catabolism. In polarized cell models, FcRn in the plasma membrane is predominantly at the basolateral surface. This distribution depends on two signals that overlap endocytosis signals. One of these signals resembles a YXXPhi motif, but with a tryptophan in place of the critical tyrosine residue; the other is a DDXXXLL signal. We examined the effects of mutations in and around these signals on the basolateral targeting of rat FcRn in rat inner medullary collecting duct cells. We also studied a second acidic cluster, Glu331/Glu333, some distance from either endocytosis signal. Some amino acid substitutions in the W-2 and W+3 positions disrupted the tryptophan-based basolateral-targeting signal without impairing its function in endocytosis. The tryptophan-based basolateral targeting and endocytosis signals are thus distinct but overlapping, as has been seen for collinear tyrosine-based signals. Surprisingly, the tryptophan-based basolateral-targeting signal required the aspartate pair of the dileucine-based signal. This acidic cluster, separated by two amino acids from the Phi residue of the tryptophan signal, is therefore a component of both of the basolateral-targeting signals. The acidic cluster Glu-331/Glu333 was not required for basolateral targeting, but its replacement reduced endocytosis.

Signals for sorting of transmembrane proteins to endosomes and lysosomes.

Sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present within the cytosolic domains of the proteins. Most signals consist of short, linear sequences of amino acid residues. Some signals are referred to as tyrosine-based sorting signals and conform to the NPXY or YXXO consensus motifs. Other signals known as dileucine-based signals fit [DE]XXXL[LI] or DXXLL consensus motifs. All of these signals are recognized by components of protein coats peripherally associated with the cytosolic face of membranes. YXXO and [DE]XXXL[LI] signals are recognized with characteristic fine specificity by the adaptor protein (AP) complexes AP-1, AP-2, AP-3, and AP-4, whereas DXXLL signals are recognized by another family of adaptors known as GGAs. Several proteins, including clathrin, AP-2, and Dab2, have been proposed to function as recognition proteins for NPXY signals. YXXO and DXXLL signals bind in an extended conformation to the mu2 subunit of AP-2 and the VHS domain of the GGAs, respectively. Phosphorylation events regulate signal recognition. In addition to peptide motifs, ubiquitination of cytosolic lysine residues also serves as a signal for sorting at various stages of the endosomal-lysosomal system. Conjugated ubiquitin is recognized by UIM, UBA, or UBC domains present within many components of the internalization and lysosomal targeting machinery. This complex array of signals and recognition proteins ensures the dynamic but accurate distribution of transmembrane proteins to different compartments of the endosomal-lysosomal system.

Tryptophan- and Dileucine-based Endocytosis Signals in the Neonatal Fc Receptor

The neonatal Fc receptor, FcRn, transports immunoglobulin G across intestinal cells in suckling rats. FcRn enters these cells by endocytosis and is present on the apical and basolateral surfaces. We investigated the roles of aromatic amino acids and a dileucine motif in the cytoplasmic domain of rat FcRn. We expressed mutant FcRn in which alanine replaced Trp-311, Leu-322, and Leu-323, or Phe-340 in the inner medullary collecting duct cell line IMCD. Individual replacement of the aromatic amino acids or the dileucine motif only partially blocked endocytosis of (125)I-Fc, whereas uptake by FcRn containing alanine residues in place of both Trp-311 and the dileucine motif was reduced to the level obtained with the tailless receptor. Leu-314 was required for the function of the tryptophan-based endocytosis signal, and Asp-317 and Asp-318 were required for the dileucine-based signal. Nonvectorial delivery of newly synthesized FcRn to the two cell surfaces was unaffected by loss of the endocytosis signals. However, the steady-state distribution of endocytosis mutants was predominantly apical, unlike wild-type FcRn, which was predominantly basolateral. This shift appeared to arise because the loss of endocytosis signals inhibited apical to basolateral transcytosis of FcRn more than basolateral to apical transcytosis.

Trafficking of major histocompatibility complex class II molecules in human B-lymphoblasts deficient in the AP-3 adaptor complex

The major histocompatibility complex class II subunits (MHC-II) α and β assemble with the invariant chain (Ii) in the endoplasmic reticulum and are transported to endosomal–lysosomal organelles known as MHC class II compartments (MIICs). Although it has been shown that two dileucine-based signals in the cytosolic tail of Ii, as well as a dileucine-based signal in the tail of the β chain mediate sorting to MIICs, the molecular mechanisms by which αβIi complexes are sorted have yet to be resolved fully. The AP-3 adaptor complex stands out as a particularly good candidate for mediating this targeting because: (i) it has a proven role in the trafficking of membrane proteins to lysosome-related organelles; and (ii) it has the ability to interact with dileucine-based signals in vitro. To investigate the potential role of AP-3 in transport of MHC-II to MIICs, we have examined MHC-II trafficking in human B-lymphoblast lines from patients with Hermansky–Pudlak syndrome type 2 (HPS-2), which are deficient in the AP-3 complex. Pulse-chase analyses revealed no significant alteration in the kinetics of synthesis and degradation of either MHC-II subunits or Ii. Moreover, we observed neither impairment of the formation of compact SDS-resistant αβ dimers, nor delay in the appearance of a conformational epitope indicative of a mature, Ii-free αβ dimer. Finally, we demonstrated that in HPS-2 patients’ cells, there was no delay in the expression of the αβ dimers on the cell surface. Thus, AP-3 does not seem to be essential for normal trafficking of MHC-II. These findings have important implications for HPS-2 patients, because they suggest that the recurrent bacterial infections suffered by these patients are not likely due to impaired antigen processing and presentation by MHC-II.

Related leucine-based cytoplasmic targeting signals in invariant chain and major histocompatibility complex class II molecules control endocytic presentation of distinct determinants in a single protein.

Leucine-based signals in the cytoplasmic tail of invariant chain (Ii) control targeting of newly synthesized major histocompatibility complex class II molecules to the endocytic pathway for acquisition of antigenic peptides. Some protein determinants, however, do not require Ii for effective class II presentation, although endocytic processing is still necessary. Here we demonstrate that a dileucine-based signal in the cytoplasmic tail of the class II beta chain is critical for this Ii-independent presentation. Elimination or mutation of this signal reduces the rate of re-entry of mature surface class II molecules into the endocytic pathway. Antigen presentation controlled by this signal does not require newly synthesized class II molecules and appears to involve determinants requiring only limited proteolysis for exposure, whereas the opposite is true for li-dependent determinants. This demonstrates that related leucine-based trafficking signals in li and class II control the functional presentation of protein determinants with distinct processing requirements, suggesting that the peptide binding sites of newly synthesized versus mature class II molecules are made available for antigen binding in distinct endocytic compartments under the control of these homologous cytoplasmic signals. This permits capture of protein fragments produced optimally under distinct conditions of pH and proteolytic activity.