Invited Symposium: Intracellular Traffic of Organelles
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Introduction
The body's ability to maintain its fluid and electrolyte balance is due in large part to the structural organization of its epithelial cells. The plasma membranes of transporting epithelial cells are divided into apical and basolateral surfaces, which comprise two morphologically and biochemically distinct domains separated by occluding junctions. These two membrane domains are distinguished by markedly different protein components that mediate the majority of their characteristic functions [1,2,3]. It is the asymmetric apportioning of ion channels, co-transporters, counter-transporters and pumps among these two surfaces that determines the direction and magnitude of the fluxes maintained by a given epithelium.
The generation and maintenance of these differentiated plasmalemmal domains requires the cell to possess machinery capable of discriminating among newly synthesized membrane proteins. Information embedded in some aspect of these proteins' architecture must serve as sorting signals which, when interpreted by the cellular sorting apparatus, specify their appropriate subcellular destinations [4]. The mechanisms and structural correlates of this sorting function have been the subject of intense research.
Recently, it has become clear that the processes which orchestrate the polarized distributions of transport proteins may also regulate their function. Several transport proteins are not constitutive denizens of a particular cell surface domain. Instead, these proteins commute between the plasmalemma and an intracellular storage compartment. In response to changes in intracellular second messenger concentrations, transporters are either inserted into or retrieved from the plasmalemma. By manipulating the surface populations of selected transport proteins, epithelial cells can precisely modulate their physiologic properties. The signals and pathways which are responsible for this regulated trafficking may be closely related to those which determine transport proteins' polarized localizations.
Signals for Polarized Sorting
Identified signals
Several classes of membrane protein sorting signals have been identified and characterized. In many epithelial cell types, attachment to the membrane through a glycosylphosphatidyl inositol (GPI) linkage is sufficient to ensure apical targetting [5-8]. Extensive mutagenesis analysis of the cytoplasmic tail of the vesicular stomatitis virus (VSV) G protein reveals that sequences of the form Tyr-X-X-Aliphatic are capable of mediating basolateral targetting [9]. Sequences resembling this motif also appear to be competant to direct sorting to a number of subcellular organelles, including endosomes, lysosomes and the trans Golgi network (TGN) [9]. Both the low density lipoprotein (LDL) receptor [10,11] and the polymeric immunoglobulin (pIgA) receptor [12,13] manifest distinct basolateral sorting signals which include tyrosine residues but do not conform to the design detected in the studies of the VSV G protein. Finally, sequences which incorporate tandem leucine residues, or di-leucine motifs, are also substrates for basolateral sorting [10].
Polytopic transport proteins
While the outline presented in the preceding paragraph summarizes an enormous amount of difficult and elegant experimental work, it is by no means exhaustive. It is clear that many, and perhaps even the majority of proteins which are distributed with polarity in epithelial cells achieve their anisotropic distributions through the influence of as yet unelucidated sorting signals. The large families of polypeptides which participate in epithelial transport certainly fall into this category. Until recently, no clearly defined signals have been linked to the polarized sorting of pumps, channels or carriers. The molecular dissection of sorting signals has largely been carried out on polypeptides whose associations with the bilayer are through GPI tails or via single trans-membrane spans. The size and complexity of polytopic membrane proteins hamper studies of the mechanisms responsible for their sorting.
Fortunately, many transport proteins belong to large families, some of whose members are targetted to distinct subcellular destinations while nevertheless sharing substantial levels of sequence identity. This property is exemplified by the isoforms of the Na,H exchnager (NHE). While NHE1 is expressed basolaterally in numerous epithelial cell types, the highly homologous NHE3 protein is restricted to the apical brush border membranes of proximal tubule cells and intestinal enterocytes, where it serves as a critical component of Na, HCO3 resorption [14-16]. Similarly, the renal isorforms of the Na,K,Cl cotrantsporter carry these three ions across the apical plasma membranes of the cells of the thick ascending limb of Henle's loop [17,18]. Although the isoform of this protein expressed in the secretory epithelial cells of the intestinal and respiratory tracts is closely related to the renal cotransporter, it accumulates at the basolateral plasma membrane [19]. Comparable distributional diversity can also be appreciated among members of transporter families expressed by transfection in polarized epithelial cells in culture. Each of the five proteins which constitute the facilitated glucose carrier family (GLUT1-5) have been exogenously expressed in the MDCK line of polarized canine kidney epithelial cells [20]. The GLUT1 and GLUT2 isoforms accumulate at the basolateral plasmalemma, whereas GLUT3 and GLUT5 behave as apical polypeptides. GLUT4, which is sorted to intracellular storage vesicles in insulin-responsive muscle and adipose cells, is also retained in an intracellular compartment in MDCK cells.
Chimera Studies
Recent studies have exploited the existence of such homologous transport protein families by creating and expressing molecular chimeras composed of complimentary portions two differnetially targetted family members. By analyzing the subcellular distributions of the resultant hybrid transporters in transfected epithelial cells, it has been possible to identify domains within the parent transport proteins' primary sequences which contribute to their sorting behaviors. It is clear from this line of research that transport proteins which manifest the same subcellular localizations can employ very different classes of sorting signals to achieve their identical distributions.
Neurotransmitter transport systems
Epithelial cells of the renal medulla import betaine from the plasma to serve as a cytoplasmic osmolyte, which helps to protect them from the extremely high osmolarity of the extracelluar fluid in the medullary interstitium [21]. This uptake is mediated by the Na,Cl-dependent betaine transporter (BET), a basolateral membrane protein whose hydropathy plot predicts twelve membrane spanning helices [22]. Molecular cloning studies have revealed that BET is closely related to the neuronal transport proteins which retrieve the neurotransmitter g-aminobutyric acid (GABA) from the synaptic space and return it to presynaptic axon terminals. This relationship is functional as well as physical, since the KM for GABA uptake by BET is actually lower than its KM for betaine transport. When expressed in MDCK cells, the GAT1 and GAT3 isoforms (which are endogenously expressed only in nervous tissue in situ) exhibit exclusively apical distributions [23,24]. The GAT2 isoform, which is normally expressed in epithelial as well as in neuronal cell types, is concentrated at the basolateral surfaces of transfected MDCK cells [24].
Several GAT1/BET and GAT2/GAT3 chimeras have been produced and their sorting behaviors characterized. This work reveals that the C-terminal cytoplasmic tails of BET and GAT-2 embody strong basolateral sorting information [25; and Muth TR, Caplan MJ, unpublished data]. Transfer of the last 20 amino acids of GAT2 to the C-terminus of GAT3 is sufficient to redirect this protein to the basolateral membrane [50]. Similarly, appending the C-terminal tail of BET to the normally apical nerve growth factor (NGF) receptor causes this protein to become an occupant of the basolateral plasmalemma [25]. Deletion studies demonstrate that the corresponding C-terminus of GAT3 contains the information which determines this protein's apical disposition. In light of these observations, it is surprising that the signal responsible for the apical sorting of GAT1 does not appear to reside near this protein's C-terminus [25].
Other neurotransmitter reuptake systems also appear to incorporate non C-terminal sorting information. The Na and Cl dependent co-transporters responsible for the presynaptic reuptake of serotonin (SERT) and norepinepherine (NET) behave as basolateral proteins when expressed in MDCK cells. In contrast, the dopamine transport system (DAT) is predominantly apical in this cell type [26]. Preliminary studies of NET/DAT chimeras suggest that sorting information resides within the N-terminal 1/3 of these proteins' primary sequences [Gu H and Rudnick G, personal communication]. Although the precise amino acid motifs responsible for BET, GAT, DAT, NET and SERT sorting have yet to be elucidated, it seems clear that multiple different classes of sorting signals, present individually or in combination, are required to encode these polypeptides' polarized distributions.
P-type ion pumps Multiple signals also appear to cooperate in the proper targetting of two members of the P-type family of ion transporting ATPases. The Na,K-ATPase is a component of the basolateral plasmalemma in most polarized epithelial cell types [27]. Its close cousin, the gastric H,K-ATPase, is sorted to the apical membrane and to a pre-apical intracellular vesicular storage compartment in its native gastric parietal cells [28]. Both pumps are composed of alpha sunbunits predicted to span the membrane ten times and beta subunits which appear to cross the membrane once in a Type II orientation [29,30]. Despite their distinct subcellular localizations, these pumps' alpha subunits are ~65% identical, while their beta subunits exhibit ~35% sequence identitiy. Chimera studies performed on these subunit polypeptides reveal that each of them encodes a distinct sorting signal [31].
Information sufficient to sort these pumps to the apical and basolateral membranes of polarized cultured LLC-PK1 epithelial cells resides within the fourth transmembrane domains (TM4) of their alpha subunits [Dunbar LA, Caplan MJ, unpublished data]. Comparison of the sequences of the TM4 domains of the Na,K and H,K-ATPase alpha subunits reveals only eight non-identical amino acids. Together with the influenza virus neuraminidase [32], these pumps appear to define a new family of plasmalemmal proteins which are sorted by virtue of membrane spanning sequences. It has been proposed that co-clustering with glycosphingolipids (GSLs) may mediate the segregation and subsequent apical sorting of GPI-linked and transmembrane proteins as they traverse the trans Golgi network (TGN) [33,34]. The TM4 sequences may thus function in sorting by defining whether the pumps can partition into GSL-rich membrane domains. This concept receives support from recent experiments in which the initial targetting of the Na,K-ATPase was monitored in MDCK cells which had been treated with fumonisin, a drug which blocks the synthesis of GSLs [35]. Whereas the pump was vectorially targetted to the basolateral surface in untreated cells, it was randomly delivered to both surface domains in the presence of the compound. It seems quite possible, therefore, that interactions between GSLs and TM4 are sufficient to effect pump sorting.
Regulation of Transporter Function by Endo and Exocytosis
A growing list of epithelial transport processes are modulated by the regulated surface delivery or retrieval of a channel, carrier or pump. Control of renal acid secretion appears to involve a redistribution of the vacuolar-type proton pump between endosomes and the plasma membrane in intercalated cells [36]. Activation of water resorption in the collecting tubule by anti-diuretic hormone (ADH) stimulates the insertion and recycling of the aquaporin II water channel [37]. Renal phosphate resorption via the type II Na/Pi cotransport system is inhibited by parathyroid hormone, which induces the specific internalization of this protein from the membrane of the proximal tubule brush border [38]. The surface expression of the epithelial Na channel (ENaC) appears to be modulated by regulated internalization or degradation [39]. Sequences in the cytosolic tails of the ENaC subunit polypeptides specify the down-regulation of the channel complex. Disruption of these sequences leads to constitutive channel activity at the plasmalemma [40]. The molecular interactions which govern this trafficking are beginning to be elucidated [41].
Transporter-induced membrane cycling
Recent studies have led to the surprising conclusion that certain transport proteins may themselves induce the expression of regulated endo- and exocytic pathways. Although LLC-PK1 cells endogenously express ADH receptors, this hormone normally has no effect on their rate of endocytosis. In LLC-PK1 cells transfected with the cDNA encoding aquaporin II, however, ADH stimulates a dramatic increase in fluid phase uptake from the apical surface [42, 43]. Similarly, cells expressing the cystic fibrosis transmembrane conductance regulator (CFTR) respond to elevations in cytosolic cAMP with increased exocytosis and decreased fluid phase endocytosis [44,45]. Presumably, both of these polypeptides are able to associate with cellular proteins that regulate membrane fusion and fission. Identifying these proteins and elaborating their relationship to specific transporters and transport processes will doubtless be the focus of a great deal of research in the near future.
Signals for regulated delivery
Some insight into the nature of the signals which govern these regulated trafficking events has been gained from studies of the gastric H,K-ATPase. This pump comprises yet another example of a transport protein which cycles between the cell surface and an intracellular population of vesicles. Stimulation of acid secretion initiates the fusion of these vesicles with the apical surfaces of gastric parietal cells, resulting in the incorporation of the H,K-ATPase into the apical plasmalemma [46,47]. Cessation of acid secretion is brought about, in part, by the retrieval of the pumps from the cell surface to their storage compartment. An examination of the cytoplasmic tail of the gastric H,K-ATPase beta subunit reveals the presence of a four amino acid sequence motif containing a tyrosine residue, which is highly homologous to well characterized tyrosine-based endocytosis signals [48,49]. To test the possibility that this sequence mediates the regulated internalization of H,K pump, the tyrosine residue was mutated to an alanine and the altered beta subunit was expressed in transgenic mice. Mice which produce the mutant protein continuously secrete gastric acid in a stimulus-independent fashion [51]. All of these animals develop gastric ulcers, presumably as a consequence of unremitting hyperacidity. Thus, while the alpha subunit of the H,K-ATPase specifies the pump's apical localization, the beta subunit's signal is responsible for governing the duration of the pump's presence at the apical surface.
Conclusions
The molecular natures of the signals which ensure the proper sorting and targetting of ion transport proteins in polarized epithelial cells have yet to be fully elucidated. The examples of the H,K-ATPase and the GABA transporter isoforms demonstrate that several different classes of signals must exist. This multiplicity of signals may allow individual epithelial cell types to differentially distribute particular transport proteins in order to accomodate distinct physiologic functions. Finally, individual transport proteins or transport protein complexes may embody more than one trafficking signal. Presumably, this multiplicity of signals participates not only in the transporter's polarized sorting, but also in regulating its presence at the plasmalemma.
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