The p58-positive pre-Golgi intermediates consist of distinct subpopulations of particles that show differential binding of COPI and COPII coats and contain vacuolar H+-ATPase

We have studied the structural and functional properties of the pre-Golgi i ntermediate compartment (IC) in normal rat kidney cells using analytical ce ll fractionation with p58 as the principal marker. The sedimentation profil e (sediterm) of p58, obtained by analytical differential centrifugation, re vealed in steady-state cells the presence of two main populations of IC ele ments whose average sedimentation coefficients, (s) over bar (H)=1150+/-58S ('heavy') and (s) over bar (L)=158+/-8S ('light'), differed from the (s) o ver bar -values obtained for elements of the rough and smooth endoplasmic r eticulum, High resolution analysis of these subpopulations in equilibrium d ensity gradients further revealed that the large difference in their (s) ov er bar -values was mainly due to particle size. The 'light' particle popula tion contained the bulk of COPI and COPII coats, and redistribution of p58 to these particles was observed in transport-arrested cells, showing that t he two types of elements are also compositionally distinct and have functio nal counterparts in intact cells. Using a specific antibody against the 16 kDa proteolipid subunit of the vacuolar H+-ATPase, an enrichment of the V-0 domain of the ATPase was observed in the p58-positive IC elements. Interes tingly, these elements could contain both COPI and COPII coats and their de nsity distribution was markedly affected by GTP gammaS, Together with morph ological observations, these results demonstrate that, in addition to clust ers of small tubules and vesicles, the IC also consists of large-sized stru ctures and corroborate the proposal that the IC elements contain an active vacuolar H+-ATPase.