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Abstract
Aquaporin-3 (AQP3) and aquaporin-4 (AQP4) are homologous proteins expressed in the basolateral plasma membrane of kidney collecting duct principal cells, where they mediate the exit pathway for apically reabsorbed water. Although both proteins are localized to the same plasma membrane domain, it is unknown if they are sorted together in the Golgi, or arrive in the same or different vesicles at the plasma membrane. We addressed these questions using high resolution deconvolution imaging, spinning disk and laser scanning confocal microscopy of cells expressing AQP3 and AQP4. AQP3 and AQP4 were observed mostly in separate post-Golgi carriers, and spinning disk microscopy showed that most of AQP3 and AQP4 were delivered to the plasma membrane in separate vesicles. In contrast, VSV-G and LDL-R, two well-charcterized basolateral proteins, co-localized to a high degree in the same post-Golgi carriers, indicating that the differential sorting of AQP3 and AQP4 is specific and regulated. Significantly, a chimeric AQP3 containing the AQP4 cytoplasmic tails co-localized with AQP4 in post-Golgi vesicles. These results indicate that AQP3 and AQP4 are separated into different post-Golgi carriers based on different cytoplasmic domain sorting signals, and are then delivered separately to the plasma membrane.
Highlights
Polarized epithelia separate different biological compartments in an organism and transfer ions and solutes between those compartments
AQP4 sorting in the trans-Golgi Network (TGN) Sorting of AQP3 and AQP4 in the TGN was analyzed in MDCK cells stably co-expressing AQP3-EGFP and OrangeAQP4, and by immunofluorescence of MDCK cells transiently coexpressing untagged AQP3 and AQP4
We identified many examples in which AQP3-EGFP and Orange-AQP4 appeared to be separated into different regions of the TGN (Figure 1), and fewer areas in which they appeared to overlap (Figure 1; yellow, overlay; see Figure S3 for an example captured by spinning disk microscopy, Figure S4 for z-view, and Figure S5 for time 0 min)
Summary
Polarized epithelia separate different biological compartments in an organism and transfer ions and solutes between those compartments. Specific channels and transporters are localized to either the apical or the basolateral membrane domain and regulate vectorial transport of water, ions and nutrients between these compartments (for review see [1]). To maintain functional cell polarity, newly-synthesized plasma membrane proteins are delivered to the correct plasma membrane domain. The number and type of newly synthesized proteins delivered to the plasma membrane is regulated to maintain homeostasis in response to physiological challenges. Considerable evidence has accumulated that in most cells apical and basolateral proteins are sorted from each other in the TGN [2] from which they exit in separate carriers and are transported to their target membrane (reviewed in [3]). Apical proteins are thought to be sorted in the trans-Golgi Network (TGN) via lipid rafts and delivered directly to the apical membrane (reviewed in [3,5])
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