T1-T1 interactions occur in ER membranes while nascent Kv peptides are still attached to ribosomes

For voltage-gated K+ channels (Kv), it is not clear at which stage during b iosynthesis in the endoplasmic reticulum (ER) oligomerization occurs, speci fically whether it can begin while nascent peptide chains of individual sub units are still attached to ribosomes. Kv channels possess a T1-recognition domain in the NH2-terminus, which confers subfamily specificity for inters ubunit assembly and forms a tetramer. Using pairs of cysteines engineered i nto the T1-TI interface and cross-linking methods, we show that specific re sidues in the T1-T1 interface of different Kv1.3 subunits come into close p roximity in the ER, both in microsomal membranes and in Xenopus oocytes. Fu rthermore, using translocation intermediates containing pairs of engineered cysteines in the TI interface, we demonstrate that specific residues in th e folded TI domain interface can approach within 2 Angstrom of each other a nd form tetramers while the nascent Kv1.3 peptides are still attached to ri bosomes and have translocated across the membrane. ER membranes are require d for this interaction, and T1-T1 interactions occur inter-polysomally. Thu s, folding of the T1 domain and intersubunit interaction may represent the first assembly event in channel formation.