Invited Symposium: Intracellular Traffic of Organelles
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 . 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
Polytopic transport proteins
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 . 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 . 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.
Neurotransmitter transport systems
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 . 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 . 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 .
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 . 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 . 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 . 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 .
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 , 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 . 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 . Activation of water resorption in the collecting tubule by anti-diuretic hormone (ADH) stimulates the insertion and recycling of the aquaporin II water channel . 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 . The surface expression of the epithelial Na channel (ENaC) appears to be modulated by regulated internalization or degradation . 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 . The molecular interactions which govern this trafficking are beginning to be elucidated .
Transporter-induced membrane cycling
Signals for regulated delivery
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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|Muth, TR; Ahn, J; (1998). Sorting of GABA Transporters in Polarized Cells. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/klip/muth0302/index.html|
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