POOR LOADING OF MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-II MOLECULES WITH ENDOGENOUSLY SYNTHESIZED SHORT PEPTIDES IN THE ABSENCE OF INVARIANT CHAINS

In normal antigen-presenting cells, newly synthesized major histocompa tibility complex (MHC) class II molecules associate with the invariant chain (Ii) glycoprotein in the endoplasmic reticulum (ER). They are l oaded with peptides only after proteolytic removal of the Ii in post-G olgi endocytic vesicles. Since the Ii inhibits peptide binding to MHC class II molecules, this association could protect MHC class II molecu les from being loaded with endogenous peptides early after biosynthesi s. If this were an important function of the Ii in vivo, MHC class II molecules synthesized in cells lacking the Ii should be loaded efficie ntly with short endogenous peptides in the ER; such peptides are known to be present there due to TAP-mediated import from the cytosol. To e xamine this possibility, we have studied peptide loading in HeLa trans fectants expressing murine H-2A(k) MHC class II molecules either alone or together with an excess of Ii. Endogenous peptides could readily b e extracted from conformationally intact A(k) alpha beta dimers of bio synthetically labeled Ii(+) cells, whereas peptide loading was greatly (> 95%) diminished in the absence of Ii. Significant amounts of sodiu m dodecyl sulfate-(SDS) stable 55-kDa peptide: A(k) complexes were onl y found in the Ii(+) transfectants. In the absence of Ii, the MHC clas s II molecules instead formed stable complexes with long (20 and 50 kD a) polypeptides. Known Ak-binding peptides bound stably to A(k) molecu les on Ii(-) cells, could be co-purified with them, and were resistant to release in SDS, suggesting that poor recovery of endogenous peptid es was not due to decreased stability of Ak: peptide complexes in the absence of Ii. We conclude that protection of MHC class II molecules f rom endogenous short peptides does not appear to be a quantitatively i mportant function of the Ii molecule, because peptide loading is ineff icient in its absence.