Our laboratory studies the distribution of phospholipids between the two leaflets of the plasma membrane lipid bilayer. In all animal cells, this distribution is asymmetric, i.e., the phospholipid composition of the two leaflets is different. One of the puzzles posed by lipid asymmetry is the mechanism by which asymmetry is generated. This distribution is generated by enzymes that actively transport phospholipids from one leaflet to the other, at the expense of ATP hydrolysis. These enzymes are members of a previously unknown subfamily of P-type ATPases, with five members in yeast and three times that number in mammals. Recently, it was discovered that these proteins are associated with subunits, proteins from the CDC50 family of transmembrane protein, with three members in both yeast and mammals. We have generated GFP-tagged constructs of several transporter genes in the mammalian family to permit investigation of the cell biology of these transporters.
Localization of GFP-tagged transporters in mammalian cells:
Jiovani Visaya '05
It is unlikely that all of the members of the P4 subfamily of amphipath transporters reside in the same subcellular compartment. As a first step to localizing the various transporters, GFP tags were added the C-termini of the proteins, and the resulting constructs then used to generate stable transfectants expressing the labeled transporters. Lines expressing the fluorescent tag at differing levels were identified and cloned, and then examined by confocal microscopy. One interesting phenotype was observed with transporter 1b, or ATP8A2. Immediately after being split (during exponential growth), the transporter moved from the ER to small cytoplasmic vesicles (Fig 10). As cells reached confluence, however, the label transferred to a few large cytoplasmic inclusions (Fig 11).
Fig 10 Fig 11
BHK cells stably transfected with ATP8A2-GFP at 3 days (Fig 10) and 7 days (Fig 11) after replating
The transfer of the transporter to inclusion bodies is observed with this and other members of the P4 ATPase family suggests that, while the initial targeting of the protein might be physiologically appropriate, subsequent mislocalization occurs as the protein accumulates. One possible explanation for this phenomenon is the absence of corresponding amounts of the appropriate CDC50 family subunit. In yeast, deletion of the appropriate subunits for a given transporter results in failure of that transporter to exit the ER, and a similar behavior in the mammalian cells would result in the observations shown in Figures 10 and 11 . In yeast, this situation is better understood, to the extent that it is known which subunits associate with which transporters. This information is not available for the mammalian transporters, but information on this point might be deducible from observing the effects of subunit on the redistribution of the transporters in stable transfected lines. In mammals, there are three members of the CDC50 subfamily, labeled A, B, and C. In preliminary experiments, these three proteins were introduced by transient transfection into the stable lines, and the distribution of the transporter was observed after splitting. As shown in Figure 12 for the CDC50A (from mice) introduced into the same line shown in figures 10 and 11, the presence of the subunit has no effect on the initial distribution of transporter in multiple small cytoplasmic vesicles. However, at intermediate times, cells appear in which the transporter is also observed to be localized to both the plasma membrane and nuclear envelope (Figure 13). A less dramatic effect was observed with CDC50B, and no effect with CDC50C (data not shown). These experiments suggest that this approach may help define the physiogical subcellular localization for transporters; in addition, these experiments may make help elucidate which CDC50 family subunits interact with which transporters in mammals.
Fig 12 Fig 13
BHK cells stably transformed with ATP8A2-GFP and transiently transfected with murine CDC50A
at 3 days (Fig 12) and 5 days (Fig 13) after replating
Localization of GFP-tagged transporters in yeast:
Mridu Kapur '06
In principle, experiments similar to those described for mammalian cells can be carried out in yeast, with the advantages of ready genetic manipulation and high resolution transport measurements as addition tools. To this end, we investigated the distribution of GFP-tagged Drs2p (the yeast analog of ATP8A1 or ATP8A2) in yeast. Two problems immediately presented themselves. One was obtaining reasonable images of specimens in the size range of yeast. This problem was eventually solved by acquisition of the high NA 100X oil immersion objective lens. The second issue arose because of the desire to correlate localization with transport data. This experiment required transformation of transport null yeast strains, a problem eventually solved by the use of kar1-mediated yeast transformation methods.