Polarized Apical Targeting Directed by the Signal/Anchor Region of Simian Virus 5 Hemagglutinin-Neuraminidase*
- From the ‡Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35209, the§Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322, the¶Department of Cancer Biology, Comprehensive Cancer Center, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157, the ‖Howard Hughes Medical Institute and Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500, and the‡Division of Endocrinology and Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Abstract
To examine the possibility of independent cytoplasmic/transmembrane domain-based apical sorting, we have investigated paramyxovirus SV5 hemagglutinin-neuraminidase (HN), a type II membrane protein with a small N-terminal signal/anchor region. In SV5-infected Madin-Darby canine kidney (MDCK) cells, >90% of HN is found on the apical surface. We have expressed chimeric proteins in which the N terminus of HN, including its signal/anchor region, is attached to a (normally cytosolic) reporter pyruvate kinase (PK). PK itself expressed immediately downstream from a cleavable signal peptide was converted to a 58-kDa N-linked glycosylated form, which was secreted predominantly (80%) to the basolateral surface of MDCK cells. By contrast, stably expressed PK chimeras, now anchored as type II membrane proteins with either the first 48 or 72 amino acids of HN, received similar N-linked glycosylation, yet exhibited polarized transport with a preferentially (75%) apical distribution. These results suggest that the N-terminal signal/anchor region of HN contains independent sorting information for apical specific targeting in MDCK cells.
Footnotes
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↵* This work was supported by National Institutes of Health Grants DK-40344 (to P. A.) and AI-12680 (to R. W. C.).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.
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↵** Investigator of the Howard Hughes Medical Institute.
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↵§§ To whom correspondence should be addressed. Tel.: 718-430-8685; E-mail: arvan{at}aecom.yu.edu.
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↵1 The abbreviations used are: HN, hemagglutinin-neuraminidase; PK, pyruvate kinase; MDCK, Madin-Darby canine kidney; PAGE, polyacrylamide gel electrophoresis; PNGase F, peptide N-glycosidase F.
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↵2 Previous expression of the PK moiety in the lumen of the secretory pathway has established that a minor fraction of PK can undergo a second distinct N-glycosylation event at a non-consensus site (39). We found that in all MDCK clones expressing secretory PK within the first 10 passages, besides ∼70% of PK that is singly glycosylated and migrates at ∼58 kDa, an additional minor form of intracellular and released PK could be detected at ∼60 kDa. We established that this minor form represented PNGase F- and endo-β-N-acetylglucosaminidase H-sensitive glycosylation at a non-consensus site, in addition to the glycosylation that occurs at the consensus asparagine, identical to that which has been reported for a fraction of HN48PK (39). For secretory PK, there is a hint of this doubly glycosylated form secreted into the medium of clonal MDCK cells from early passage in the second lane of Fig. 2 B. However, as a function of passage number, expression of this additionally glycosylated form diminished to below the limits of detection in our pulse-labeling experiments. We should note that the presence or absence of this additional glycosylation event had no detectable effect on the routing of secretory PK to the basolateral surface.
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↵3 R. A. Lamb, unpublished results.
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- Received July 22, 1997.
- Revision received August 28, 1997.
