THE 47-KD LENS-SPECIFIC PROTEIN PHAKININ IS A TAILLESS INTERMEDIATE FILAMENT PROTEIN AND AN ASSEMBLY PARTNER OF FILENSIN

In previous studies we have characterized a lens-specific intermediate filament (IF) protein, termed filensin. Filensin does not self-assemb le into regular IFs but is known to associate with another 47-kD lens- specific protein which has been suggested to represent its assembly pa rtner. To address this possibility, we cloned and sequenced the cDNA c oding for the bovine 47-kD protein which we have termed phakinin (from the greek phialphakappaomicronsigma = phakos = lens). The predicted s equence comprises 406 amino acids and shows significant similarity (31 .3% identity over 358 residues) to type I cytokeratins. Phakinin posse sses a 95-residue, non-helical domain (head) and a 311 amino acid long alpha-helical domain punctuated with heptad repeats (rod). Similar to cytokeratin 19, phakinin lacks a COOH-terminal tail domain and it the refore represents the second known example of a naturally tailless IF protein. Confocal microscopy on frozen lens sections reveals that phak inin colocalizes with filensin and is distributed along the periphery of the lens fiber cells. Quantitative immunoblotting with whole lens f iber cell preparations and fractions of washed lens membranes suggest that the natural stoichiometry of phakinin to filensin is approximatel y 3:1. Under in vitro conditions, phakinin self-assembles into metasta ble filamentous structures which tend to aggregate into thick bundles. However, mixing of phakinin and filensin at an optimal ratio of 3:1 y ields stable 10-nm filaments which have a smooth surface and are ultra structurally indistinguishable from ''mainstream'' IFs. Immunolabeling with specific antibodies shows that these filaments represent phakini n/filensin heteropolymers. Despite its homology to the cytokeratins, p hakinin does not coassemble with acidic (type I), or basic (type II) c ytokeratins. From these data we conclude that filensin and phakinin ar e obligate heteropolymers which constitute a new membrane-associated, lens-specific filament system related to, but distinct from the known classes of IFs.