Polarized Apical Targeting Directed by the Signal/Anchor Region of Simian Virus 5 Hemagglutinin-Neuraminidase

Abstract

To examine the possibility of independent cytoplasmic/transmembrane domain-based apical sorting, we have investigated paramyxovirus SV5 hemagglutininneuraminidase (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 Nterminal signal/anchor region of HN contains independent sorting information for apical specific targeting in MDCK cells.

The Transmembrane Domain of the Respiratory Syncytial Virus F Protein Is an Orientation-Independent Apical Plasma Membrane Sorting Sequence

“…Some reports suggested that glycosylation of the ED of type I glycoproteins determines their apical transport to the plasma membrane (13,19,24,30). However, other reports suggest that apical membrane targeting depends on the TMD of type I membrane proteins (14,17,28). Finally, some studies have indicated that CT residues are necessary for regulating transport between the Golgi and the plasma membrane in nonpolarized cells (25,33).…”

Raft-mediated Trafficking of Apical Resident Proteins Occurs in Both Direct and Transcytotic Pathways in Polarized Hepatic Cells: Role of Distinct Lipid Microdomains

“…In the majority of epithelial cells studied, such a mechanism has been shown for all GPIanchored proteins (Lisanti et al, 1988(Lisanti et al, , 1989Brown et al, 1989;Soole et al, 1995;Kenworthy and Edidin, 1998), and some TMD proteins (Kundu et al, 1996;Huang et al, 1997;Lin et al, 1998). However, in hepatocytes, transcytotic delivery via the basolateral membrane has been proposed to represent the major pathway for delivery of resident apical proteins (Schell et al, 1992;Maurice et al, 1994;Ihrke et al, 1998).…”

“…Liquid-ordered sphingolipid/cholesterol-enriched domains, also known as "rafts," seem instrumental in targeting of apical proteins (Simons and Ikonen, 1997;Ikonen, 2001). Sorting signals are provided by their glycosylphosphatidylinositol (GPI)-membrane anchor (Brown et al, 1989;Lisanti et al, 1989Lisanti et al, , 1990 or transmembrane domain (TMD) (Kundu et al, 1996;Huang et al, 1997;Lin et al, 1998). These sorting principles in polarized trafficking have been largely derived in Madin-Darby canine kidney (MDCK) cells.…”

Characteristics and Structural Requirements of Apical Sorting of the Rat Growth Hormone through the O-Glycosylated Stalk Region of Intestinal Sucrase-isomaltase

“…On the other hand, the TM of SI is a critical component in the overall sorting event of SI, and its presence constitutes an absolute requirement for SI to associate with lipid rafts and to be ultimately sorted to the apical membrane (19). Nevertheless and unlike the TM of certain integral viral protein, such as the influenza HA (12,37), neuraminidase (38), and the envelope glycoprotein of simian virus (39), the TM of SI alone is not sufficient to mediate apical targeting. In fact, an SI deletion mutant containing the TM domain, but lacking another structurally critical domain, the O-glycosylated stalk region, is sorted randomly in epithelial cells and does not acquire TX-100 detergent insolubility during its passage through the TGN (19).…”