The hypothesis upon which this application is based is that the translation- translocation of alpha1(I) procollagen chains is facilitated by a translocon (multispaning membrane glycoproteins of the endoplasmic reticulum), and endoplasmic reticulum (ER) resident molecular chaperons. Further, the association of procollagen with individual translocon proteins and a series of molecular chaperons follows the principle of successive action. Based on the studies of the applicant, candidates for this consecutive interaction with procollagen are seen to possibly include the translocon-associated protein (TRAP), translocating chain- associating membrane protein (TRAM), the mammalian homolog of SEC61p, Hsp47 (a specific collagen binding protein), Grp78, and Grp94. Translocon proteins are proposed to act to mediate the binding of ribosomes engaged in synthesizing procollagen that has been targeted to the ER membrane by the signal recognition particle and its receptor. As translocation of the nascent chains proceeds, they are shielded from the hydrophobic core of the membrane by translocon components. The nascent chains of procollagen are then handed to a successive group of molecular chaperons, Hsp47, Grp78, and Grp94, that facilitate the translation-translocation process by reducing incorrect folding, thus insuring that translation-translocation occur with high fidelity. This cascade of protein-protein interactions ensures, in ligament cells, the constitutive synthesis of procollagen during periods of normalcy and stress. This hypothesis will be tested by completion of the following specific aims.
Specific Aim 1 will verify that a translocon comprised of translocating chain associating membrane protein (TRAM) and SEC61p exists in the ER of ligament cells and fibroblasts that produce procollagen I.
Specific Aim 2 will identify components of the mammalian endoplasmic reticulum that bind ribosomes which translate alpha1(I) procollagen mRNA, and are associated with translocation of alpha(I) procollagen nascent chains. Specific focus will be directed to TRAM, SEC61p and HSP47.
Specific Aim 3 will attempt to prove that there is a successive association between individual translocon proteins, ER resident molecular chaperons and procollagen. Utilizing crosslinking and reconstitution methods it will be determined whether Hsp47 is the first ER resident molecular chaperone to interact with evolving procollagen chains and determine the point of procollagen synthesis that is associated with Grp78, Grp94 and other ER resident proteins.
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