Identification of the basolateral targeting determinant of a peripheral membrane protein, MacMARCKS, in polarized cells.

BACKGROUND

Although the molecular determinants that specify the targeting of transmembrane proteins to the apical or basolateral membrane domains within polarized epithelial cells have been well characterized, very little is known about the targeting of peripheral membrane proteins within these cells. MacMARCKS is a member of the MARCKS family of protein kinase C (PKC) substrates. This myristoylated protein regulates actin structure at cell membranes and is essential for the morphogenic movement of neuroepithelial cells during the formation of the neural tube.

RESULTS

MacMARCKS was specifically targeted to sites of cell-cell contact in the basolateral domain of polarized Madin-Darby canine kidney (MDCK) epithelial cells and was displaced from this location upon activation of PKC. We defined the basolateral targeting determinant of MacMARCKS to be the effector domain, a basic region spanning 24 amino acids and containing the PKC phosphorylation sites as well as binding sites for calmodulin and actin. This domain, in conjunction with a myristoyl moiety, was sufficient to target a non-membrane-associated protein--green fluorescent protein--specifically to the basolateral surface of polarized MDCK cells.

CONCLUSIONS

This is the first description of a specific amino acid sequence that specifies targeting of a peripheral membrane protein to the basolateral membrane in polarized epithelial cells.