Synthesis and assembly of the vacuolar H(+)-ATPase from oat seedlings

The major objectives of my dissertation research were to understand the synthesis and assembly of the vacuolar {dollar}rm Hsp{lcub}+{rcub}{dollar}-translocating ATPase (V-ATPase). V-ATPase is a large multimeric complex consisting of {dollar}rm Vsb1{dollar} and {dollar}rm Vsb0{dollar} subcomplexes. The V-ATPase pumps {dollar}rm Hsp{lcub}+{rcub}{dollar} into endomembrane vesicles to generate a {dollar}rm Hsp{lcub}+{rcub}{dollar} gradient across the membranes. This electrochemical gradient of {dollar}rm Hsp{lcub}+{rcub}{dollar} is required for many cellular functions including osmotic regulation, signal transduction, storage and release of solutes and ions. However, the synthesis and assembly of V-ATPase are poorly understood.; To determine where the V-ATPase is synthesized and assembled, endomembrane vesicles from oat roots were fractionated by a linear sucrose gradient. The subunits of both {dollar}rm Vsb1{dollar} and {dollar}rm Vsb0{dollar} complex were detected at several membrane fractions, including the ER, provacuole and vacuoles, suggesting a model in which V-ATPase is assembled at the ER and then sorted to its destination. This model is supported by immunogold labeling in which peripheral subunit B was shown to be associated with the ER. To understand the biosynthesis and assembly machinery, I have begun to identify proteins involved in subunit folding and assembly of the V-ATPase. A 64 kD polypeptide that copurified with oat V-ATPase has been identified as calnexin, an ER resident molecular chaperone. Monoclonal antibody (MAb) against calnexin coimmunoprecipitated V-ATPase subunits; while MAb against subunit A coprecipitated calnexin and BiP with V-ATPase complex. The results suggest that both calnexin and BiP act as molecular chaperones in the folding and assembly of newly synthesized V-ATPase at the ER. The role of a polypeptide immunologically related to the 100 kD subunit of many eukaryotic V-ATPase was investigated. In oat, an ER-associated 100 kD polypeptide was associated with a {dollar}{lcub}sim{rcub}250{dollar} kD {dollar}rm Vsb0{dollar} complex containing the 36 kD and 16 kD subunits, but not associated with the fully assembled and active {dollar}rm Vsb1Vsb0{dollar}-ATPase. Pulse-chase labeling and immunoprecipitation results suggest that the {dollar}rm Vsb1{dollar} is synthesized and assembled in the cytosol. The 250 kD {dollar}rm Vsb0,{dollar} complex can be assembled in vitro with the cytosolic {dollar}rm Vsb1{dollar} to form a fully assembled V-ATPase. However, the 100 kD polypeptide was not associated with the fully assembled complex. These results suggest that the 100 kD polypeptide in oat has a role in assembly but is not required for activity of V-ATPase. Thus, the folding and assembly of oat V-ATPase at the ER depends on a specific 100 kD polypeptide as well as general chaperones, like calnexin and BiP. A working model of chaperone-assisted assembly of V-ATPase is proposed.