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 biosynthesis in the endoplasmic reticulum (ER) oligomerization occurs, specifically whether it can begin while nascent peptide chains of individual subunits are still attached to ribosomes. Kv channels possess a T1-recognition domain in the NH2-terminus, which confers subfamily specificity for intersubunit assembly and forms a tetramer. Using pairs of cysteines engineered into the T1-T1 interface and cross-linking methods, we show that specific residues in the T1-T1 interface of different Kv1.3 subunits come into close proximity in the ER, both in microsomal membranes and in Xenopus oocytes. Furthermore, using translocation intermediates containing pairs of engineered cysteines in the T1 interface, we demonstrate that specific residues in the folded T1 domain interface can approach within 2 A of each other and form tetramers while the nascent Kv1.3 peptides are still attached to ribosomes and have translocated across the membrane. ER membranes are required for this interaction, and T1-T1 interactions occur inter-polysomally. Thus, folding of the T1 domain and intersubunit interaction may represent the first assembly event in channel formation.
Lu
J
J
Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6085, USA.
Robinson
J M
JM
Edwards
D
D
Deutsch
C
C
D000818:Animals; D001665:Binding Sites; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005260:Female; D051664:Kv1.3 Potassium Channel; D008861:Microsomes / Q000378:metabolism; D008958:Models, Molecular; D010446:Peptide Fragments / Q000096:biosynthesis; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012270:Ribosomes / Q000378:metabolism*; D014982:Xenopus laevis
D051664:Kv1.3 Potassium Channel; D010446:Peptide Fragments; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated