Eukaryotic CTR Copper Uptake Transporters Require Two Faces of the Third Transmembrane Domain for Helix Packing, Oligomerization, and Function

Abstract

Members of the copper uptake transporter (CTR) family from yeast, plants, and mammals including human are required for cellular uptake of the essential metal copper. Based on biochemical data, CTRs have three transmembrane domains and have been shown to oligomerize in the membrane. Among individual members of the family, there is little amino acid sequence identity, raising questions as to how these proteins adopt a common fold, oligomerize, and participate in copper transport. Using site-directed mutagenesis, tryptophan scanning, genetic complementation, subcellular localization, chemical cross-linking, and the yeast unfolded protein response, we demonstrated that at least half of the third transmembrane domain (TM3) plays a vital role in CTR structure and function. The results of our analysis showed that TM3 contains two functionally distinct faces. One face bears a highly conserved Gly-X-X-X-Gly (GG4) motif, which we showed to be essential for CTR oligomerization. Moreover, we showed that steric constraints reach past the GG4-motif itself including amino acid residues that are not conserved throughout the CTR family. A second face of TM3 contains three amino acid positions that, when mutated to tryptophan, cause predominantly abnormal localization but are still partially functional in growth complementation experiments. These mutations cluster on the face opposite to the GG4-bearing face of TM3 where they may mediate interactions with the remaining two transmembrane domains. Taken together, our data support TM3 as being buried within trimeric CTR where it plays an essential role in CTR assembly.

Posttranslational regulation of copper transporters

“…Biochemical analysis and electron microscopic crystallography revealed that hCTR1 subunits assemble as homooligomeric complexes comprising three hCTR1 subunits [7,33,34,37,39]. Homo-oligomerization may involve mutual interactions of the amino termini of hCTR1, as was suggested by yeast-two-hybrid experiments [37].…”

“…Homo-oligomerization may involve mutual interactions of the amino termini of hCTR1, as was suggested by yeast-two-hybrid experiments [37]. A cysteine residue in the carboxy terminus [36] and a Gly-Xaa 3 -Gly sequence in the second transmembrane helix [33,34] have also been implicated in hCTR1 oligomerization. Together, these data support the notion that multiple Fig.…”

“…hCTR1 was identified as a highaffinity CTR by functional complementation of a yeast strain deficient in high-affinity copper uptake [29][30][31][32]. The characterization of CTR1 as a high-affinity CTR marked the start of extensive studies on the structure, function, and cellular localization of the CTR protein family [2,6,7,[33][34][35][36][37][38][39][40][41][42]. Overexpression of hCTR1 in several cell lines results in a substantial, specific, and saturable induction of cellular copper import with a K m of approximately 1-5 lM [7,35,39].…”

“…Electron microscopy analysis of protein crystals comprising recombinant hCTR1 reconstituted in native phospholipid bilayers permitted determination of the structure of the hCTR1 oligomer at approximately 6-Å resolution. These studies revealed that hCTR1 forms compact trimeric complexes containing nine transmembrane helices [33,34]. Experiments using hCTR2 indicated that oligomerization is a general characteristic of human CTR proteins [7].…”

Les bases moléculaires de l’approvisionnement en cuivre

“…Il existe un motif composé de deux résidus glycines espacés de trois résidus (GxxxG) (mauve) à la hauteur du TM3. Ce motif, nommé GG4, est reconnu pour stabiliser l'interaction hélice-hélice à l'intérieur de la bicouche lipidique composant les membranes et permettre la multimérisation des protéines membranaires [28]. liaison au Cu 1+ , MxCxxC, également présent chez la chaperonne Atx1.…”