Hierarchy of Sorting Signals in Chimeras of Intestinal Lactase-Phlorizin Hydrolase and the Influenza Virus Hemagglutinin

Ralf Jacob,Ute Preuss,Petra Panzer,Marwan Alfalah,Stephanie Quack,Mike G Roth,Hussein Naim,Hassan Y Naim

doi:10.1074/jbc.274.12.8061

Abstract

Lactase-phlorizin hydrolase (LPH) is an apical protein in intestinal cells. The location of sorting signals in LPH was investigated by preparing a series of mutants that lacked the LPH cytoplasmic domain or had the cytoplasmic domain of LPH replaced by sequences that comprised basolateral targeting signals and overlapping internalization signals of various potency. These signals are mutants of the cytoplasmic domain of the influenza hemagglutinin (HA), which have been shown to be dominant in targeting HA to the basolateral membrane. The LPH-HA chimeras were expressed in Madin-Darby canine kidney (MDCK) and colon carcinoma (Caco-2) cells, and their transport to the cell surface was analyzed. All of the LPH mutants were targeted correctly to the apical membrane. Furthermore, the LPH-HA chimeras were internalized, indicating that the HA tails were available to interact with the cytoplasmic components of clathrin-coated pits. The introduction of a strong basolateral sorting signal into LPH was not sufficient to override the strong apical signals of the LPH external domain or transmembrane domains. These results show that basolateral sorting signals are not always dominant over apical sorting signals in proteins that contain each and suggest that sorting of basolateral from apical proteins occurs within a common compartment where competition for sorting signals can occur.

Highlights

Polarized cells such as neurons and epithelial cells maintain separate plasma membrane domains, each with a distinct protein and lipid composition, through intracellular sorting mechanisms that recognize classes of proteins and deliver them into separate vesicles for transport to the correct surface domain [1, 2]
To investigate the position and relative strength of the apical sorting signal of Lactase-phlorizin hydrolase (LPH), sorting of a tailless LPH mutant (LPHϪct) [39] and chimeric proteins made by fusing LPH external and transmembrane sequences to the short, 12-amino acid-long cytoplasmic domain of several HA mutants was studied in Madin-Darby canine kidney (MDCK) cells
The pulse-chase and sorting analyses indicate that intracellular processing and targeting of proLPHϪct in MDCK cells is similar to its wild type pro-LPH counterpart and that the cytosolic portion of pro-LPH is devoid of apical sorting signals

Summary

EXPERIMENTAL PROCEDURES

Construction of cDNA Encoding Mutant LPHϪct and Chimeras of Intestinal LPH and Influenza Virus HA—Standard recombinant DNA techniques were employed according to Sambrook et al [44]. Expression of LPH-HA chimeras in intestinal Caco-2 cells was preformed transiently on membrane filters using the calcium phosphate procedure [48]. We found that a 3-day period was sufficient for the cell layer to achieve complete polarity This was biochemically assessed by cell surface immunoprecipitation of sucrase-isomaltase, which is targeted in Caco-2 cells to the apical membrane. Caco-2 cells expressing transiently transfected LPH-HAwt or LPH-HAY543/F546 were labeled continuously for 18 h to ensure a maximum labeling of the expressed recombinant proteins. Chimeras containing the external and transmembrane domain of LPH and the cytoplasmic domain of HA are indicated as LPH-HA. Single or double mutations in the cytoplasmic tail of HA are indicated by underlined letters

Transmembrane sequence Cytoplasmic sequence

RESULTS

DISCUSSION