Multiple Sorting Signals Determine Apical Localization of a Nonglycosylated Integral Membrane Protein*
- From the Centro de Biologı́a Molecular “Severo Ochoa,” Consejo Superior de Investigaciones Cientı́ficas-Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
Abstract
In polarized cells, newly synthesized proteins are sorted in the trans-Golgi network and from there delivered to either the apical or basolateral membranes. Madin-Darby canine kidney (MDCK) cells have been widely used as a model system to study sorting determinants to the apical and basolateral surfaces. Whereas sorting signals for basolateral transmembrane proteins seem to reside in their cytoplasmic domains, apical determinants appear to reside in the N-glycans of secretory proteins or in the glycolipid tails of glycosylphosphatidylinositol-linked proteins. We show in this study that a surface-expressed form of CD3-ε, a nonglycosylated type I membrane protein, is exclusively targeted to the apical membrane in MDCK cells by a glycolipid-independent transport pathway. Deletion of the cytoplasmic tail does not affect its distribution, whereas deletion of the transmembrane domain results in secretion from both surfaces although still predominantly through the apical membrane. The transmembrane domain of CD3-ε appended to rat growth hormone, a secretory protein that lacks apical and basolateral determinants, promotes basolateral localization of the chimeric protein. However, a growth hormone chimera containing both the transmembrane and cytoplasmic domains of CD3-ε resulted in localization to the apical and basolateral membranes. These results suggest there are multiple determinants in CD3-ε that affect its distribution in polarized MDCK cells. Whereas the transmembrane domain contains a basolateral determinant, the ectodomain and the cytoplasmic domain contain apical determinants.
Footnotes
-
↵* This work was supported by Comisión Interministerial de Ciencia y Tecnologı́a, Spain, Grants PM950005, PB93-0175, and PM96-0004, Comunidad de Madrid Grants 07/047/96 and SAL AE00309/95, and grants from Fundación Rodrı́guez Pascual. The institutional support of Fundación Ramón Areces is also acknowledged.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵‡ Holder of a European Union fellowship. Present address: Dept. of Microbiology, Norman Bethune University of Medical Sciences, Changchun, Jilin 130021, China.
-
↵§ To whom correspondence should be addressed. Tel.: 34-1-3978049; Fax: 34-1-3978087.
-
↵1 The abbreviations used are: MDCK, Madin-Darby canine kidney; ER, endoplasmic reticulum; GH, growth hormone; GPI, glycosylphosphatidylinositol; PBS, phosphate-buffered saline; TM, transmembrane domain; TMC, transmembrane and cytosolic domains; MES, 4-morpholineethanesulfonic acid.
-
- Received July 1, 1997.
- Revision received September 22, 1997.
