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Identification of the basolateral targeting determinant of a peripheral membrane protein, MacMARCKS, in polarized
M M Myat1, S Chang, E Rodriguez-Boulan
1Rockefeller University, New York, New York 10021, USA.
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.
Insights
Researchers identified the specific protein region responsible for targeting peripheral membrane proteins to the basolateral domain in polarized epithelial cells. This finding advances understanding of cellular organization and protein localization.
Area of Science:
- Cell Biology
- Molecular Biology
- Epithelial Cell Biology
Background:
- While transmembrane protein targeting in polarized epithelial cells is understood, peripheral membrane protein localization remains unclear.
- MacMARCKS, a myristoylated protein kinase C (PKC) substrate, regulates actin structure and is crucial for neuroepithelial cell morphogenesis during neural tube formation.
Purpose of the Study:
- To investigate the molecular mechanisms governing the basolateral targeting of the peripheral membrane protein MacMARCKS in polarized epithelial cells.
- To identify the specific domains within MacMARCKS responsible for its localization to the basolateral membrane.
Main Methods:
- Utilized Madin-Darby canine kidney (MDCK) epithelial cells, a model system for polarized epithelial cells.
- Investigated MacMARCKS localization in response to protein kinase C (PKC) activation.
- Employed green fluorescent protein (GFP) fusion constructs to test the sufficiency of identified targeting determinants.
Main Results:
- MacMARCKS was observed to localize to the basolateral domain, specifically at sites of cell-cell contact.
- PKC activation led to the displacement of MacMARCKS from its basolateral location.
- The effector domain of MacMARCKS, a 24-amino acid basic region, was identified as the key basolateral targeting determinant.
- This effector domain, along with the myristoyl moiety, was sufficient to direct GFP to the basolateral surface of polarized MDCK cells.
Conclusions:
- This study provides the first identification of a specific amino acid sequence dictating peripheral membrane protein targeting to the basolateral membrane in polarized epithelial cells.
- The findings elucidate a novel mechanism for peripheral membrane protein localization, contributing to our understanding of epithelial cell polarity and function.