BIOSYNTHESIS OF THE GP330 44-KDA HEYMANN NEPHRITIS ANTIGENIC COMPLEX - ASSEMBLY TAKES PLACE IN THE ER

The Heymann nephritis antigenic complex (HNAC) consists of two compone nts, i.e., 1) gp330, a large glycoprotein localized in coated pits of the proximal tubule and glomerular epithelium, and 2) a 44-kDa protein which is homologous to the human alpha2-macroglobulin receptor-associ ated protein (RAP). To examine the biosynthesis and assembly of HNAC, tissue fragments prepared from collagenase-digested 1-day-old rat kidn eys were radiolabeled, and gp330 and RAP were immunoprecipitated with specific antibodies. By electron microscopy the tubule organization wa s seen to be largely intact. Results obtained on the biosynthesis of a control brush border protein, dipeptidylpeptidase IV (DPPIV), showed that tubules prepared in this manner are capable of synthesis and post translational processing of brush border membrane proteins and thus ar e suitable for short-term (<3 h) biosynthetic experiments in vitro. Re sults of pulse chase and digestion with endoglycosidase H (Endo H) ind icated that the time required for newly synthesized gp330 to mature in the endoplasmic reticulum (ER) and transit the middle Golgi compartme nts [half time (t1/2) = 90 min] was significantly longer than that of DPPIV (t1/2 = 20 min). Coprecipitation and cosedimentation (sucrose ve locity gradient centrifugation) experiments showed that gp330 associat es with RAP very early after synthesis and that the 44-kDa protein rem ains associated with gp330 during its subsequent folding, oligomerizat ion, and transport to the Golgi. These findings demonstrate that HNAC assembles in at least two steps. The first step is the association of gp330 with RAP forming a large (19.3S) heterodimer, which sediments wi th the thyroglobulin (mol wt = 669,000) standard. This step begins wit hin 30 min of synthesis and is Ca2+ dependent. The second step, which occurs >60 min after synthesis, is the formation of a larger heterooli gomer, which results in a shift in size of the complex from 19.3 to 38 .6S. Both steps occur before acquisition of Endo H resistance. These r esults indicate that HNAC consists of a large multimeric complex that is assembled in the rough ER.