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Segregation of open major histocompatibility class I conformers at the plasma
membrane and during endosomal trafficking – implications for presentation
of endocytosed antigens
Mahmutefendic H1,2, Blagojevic Zagorac G1, Ilic Tomas M1, Groettrup M3, Momburg F2, Lucin P1
1
Department of Physiology and Immunology, University of Rijeka, Faculty of Medicine, Brace Branchetta 20,
51000 Rijeka, Croatia.
2
Department of Translational Immunology, German Cancer Research Center, Im Neuenheimer Feld 460, 69120
Heidelberg, Germany
3
Division of Immunology, Department of Biology, Constance University, 78457 Konstanz, Germany
Abstract
Fully conformed major histocompatibility class I molecules are complexes of heavy chain noncovalently associated with the peptide and  2-microglobulin. Conformational change in the
extracellular domain of heavy chain leads to their disassembly and formation of open conformers, a
process that physiologically occurs in normal cells and results in their presence at the cell surface. In
this study we characterized endosomal trafficking of open conformers of a Ld MHC-I molecules in
order to examine whether conformational change in the extracellular domain of a membrane
glycoprotein determines its endosomal sorting. Open conformers segregated from their fully
conformed counterparts at the plasma membrane and in endosomes by sequestration in lipidorganized membrane environment. Consequently, open conformers constitutively internalized via
distinct clathrin-independent endocytic carriers and converged into "classical" early endosomes
together with transferrin receptor and cholera-toxin B subunit. In early endosomes, open conformers
were excluded from recycling and diverted towards late endosomes. By using siRNA silencing, we
have found that this process is, to a some extent, Rab7 dependent. After Rab7 depletion open Ld
conformers are loaded in big vesicles that are only partially EEA1+. Furthermore, due to lack of
recycling, open conformers were constitutively internalized at a higher rate than full conformed
proteins. Concanamycin A treatment prevented segregation of open conformers in vacuolar early
endosomes indicating that acidic endosomal environment and membrane composition are critical for
the maintenance of the sorting mechanism. In the absence of endosomal acidification open
conformers partitioned into lipid disordered membrane composition of early endosomes. Thus, our
data suggest for the existence of a lipid-dependent mechanism in the endosomal system that
distinguish membrane proteins based on conformation of their extracellular domain. (1)
It is an important question in which endosomal subcompartment(s) and through which
mechanism fully conformed or peptide-free, open conformers recycling through endocytic vesicles
are charged with peptides derived from endocytosed antigens. (2) To investigate into this question in
more detail we have transfected Ltk- and Balb3T3 fibroblasts with either Ld, Kb or Db and pulsed them
with long peptides (~30 a.a.) harboring T cell epitopes for presentation to respective Ld, Kb or Dbrestricted T cell hybridomas. Unexpectedly, we observed that the fibroblasts lines were able to
process the exogenously added, long peptides and presented the studied epitopes. In ongoing
experiments using siRNA-mediated silencing of GTPases (Rab5a, Rab5b, Rab7, Rab11 and Arf6)
involved in vesicular transport we will investigate whether this non-classical antigen presentation
depends on specific vesicular targeting mechanisms, is independent of proteasomes and TAP, and
whether fully conformed, recycling MHC-I molecules, or their open counterparts, are preferentially
loaded with peptides processed out of exogenous antigen. Respective results will be reported.
(1) Mahmutefendic H et al., Int. J. Biochem. Cell Biol. 43: 504-515 (2011)
(2) Tiwari et al., J. Immunol. 178: 7932-7942 (2007)