PKCzeta is required for microtubule-based motility of vesicles containing the ntcp transporter.
Intracellular trafficking regulates the abundance and therefore activity of transporters present at the plasma membrane. The transporter, Na+-taurocholate co-transporting polypeptide (ntcp), is increased at the plasma membrane upon treatment of cells with cAMP, for which microtubules (MTs) are required and the PI3K pathway and PKCzeta have been implicated. However, trafficking of ntcp on MTs has not been demonstrated directly and the regulation and intracellular localization of ntcp is not well understood. Here, we utilize in vitro and whole-cell immunofluorescence microscopy assays to demonstrate that ntcp is present on intracellular vesicles that bind MTs and move bidirectionally, using kinesin-1 and dynein. These vesicles co-localize with markers for recycling endosomes and early but not late endosomes. They frequently undergo fission, providing a mechanism for the exclusion of ntcp from late endosomes. PI(3,4,5)P3 activates PKCzeta and enhances motility of the ntcp vesicles and overcomes the partial inhibition produced by a PI3-kinase inhibitor. Specific inhibition of PKCzeta blocks the motility of ntcp-containing vesicles but has no effect on late vesicles as shown both in vitro and in living cells transfected with ntcp-GFP. These data indicate that PKCzeta is required specifically for the intracellular movement of vesicles that contain the ntcp transporter.
Sarkar
Souvik
S
Marion Bessin Liver Research Center, Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Bananis
Eustratios
E
Nath
Sangeeta
S
Anwer
M Sawkat
MS
Wolkoff
Allan W
AW
Murray
John W
JW
D000818:Animals; D004398:Dyneins / Q000378:metabolism; D004705:Endocytosis; D016547:Kinesins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D000099221:Protein Kinase C zeta
D004398:Dyneins; D016547:Kinesins; D011493:Protein Kinase C; D000099221:Protein Kinase C zeta