Basolateral protein transport in streptolysin O-permeabilized MDCK cells.
We have reconstituted polarized protein transport in streptolysin O-permeabilized MDCK cells from the TGN to the basolateral surface and to the apical surface. These transport steps are dependent on temperature, energy and exogenously supplied cytosol. Using this in vitro system we show that a whole tail peptide (WT peptide) corresponding to the cytoplasmic tail of a basolaterally sorted protein, the vesicular stomatitis virus glycoprotein (VSV G) inhibits the TGN to basolateral transport but does not affect any other transport step. Inhibition of VSV G transport to basolateral surface by WT peptide did not result in missorting of the protein to the apical surface. Mutation of the single tyrosine residue in the WT peptide reduced its inhibitory potency four- to fivefold. These results suggest that the VSV G tail physically interacts with a component of the sorting machinery. Using a cross-linking approach, we have identified proteins that associate with the cytoplasmic tail domain of VSV G. One of these polypeptides, Tin-2 (Tail interacting protein-2), associates with VSV G in the TGN, the site of protein sorting, but not in the ER nor at the cell surface. Tin-2 does not associate with apically targeted hemagglutinin. WT peptide that inhibited the basolateral transport of VSV G also inhibited the association of Tin-2 with VSV G. Together, these properties make Tin-2 a candidate basolateral sorter. The results demonstrate the usefulness of the SLO-permeabilized cell system in dissecting the sorting machinery.
Pimplikar
S W
SW
Cell Biology Programme, European Molecular Biology Laboratory, Heidelberg, Germany.
Ikonen
E
E
Simons
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D001692:Biological Transport; D002460:Cell Line; D002463:Cell Membrane Permeability; D016764:Cell Polarity; D003593:Cytoplasm / Q000378:metabolism; D004285:Dogs; D006056:Golgi Apparatus / Q000378:metabolism; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D013301:Streptolysins / Q000494:pharmacology; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D010455:Peptides; D013301:Streptolysins; D014759:Viral Envelope Proteins; C025964:streptolysin O