The vacuolar H + -ATPase (V-ATPase) is a universal component of eukaryotic organisms. It is present in the membranes of many organelles, where its proton-pumping action creates the low intravacuolar pH found, for example, in lysosomes. In addition, there are a number of differentiated cell types that have V-ATPases on their surface that contribute to the physiological functions of these cells. The V-ATPase is a multi-subunit enzyme composed of a membrane sector and a cytosolic catalytic sector. It is related to the familiar F o F " ATP synthase (F-ATPase), having the same basic architectural construction, and many of the subunits from

Electron microscopic analysis of gap-junction-like structures isolated from an arthropod (Nephrops nowegicus) and composed of a 16-kDa polypeptide, show the functionaI unit to be a star-shaped hexamer of protein arranged around a central channel which runs perpendicular to the plane of the membrane. Estimations of the molecular volume carried out on an averaged projection are consistent with a subunit mass of 16-18 kDa. Fourier transform infrared spectroscopy indicates a high a-helical content for the protein, supporting secondary-structure predictions of four transmembrane a helices/monomer. The averaged projection shows a close resemblance to a hexamer of the 16-kDa protein built on the basis of a four a-helical bundle [Finbow,

The bag cells in the abdominal ganglion of Aplysia californica control egg-laying behavior by releasing a polypeptide (ELH) during an afterdischarge of synchronous action potentials. We have used intracellular injection of Lucifer Yellow to study the morphology and interconnections of the bag cells. These neurosecretory cells are typically multipolar and their processes extend in all directions out from the bag cell clusters into the surrounding connective tissue, where they branch in a complex manner. In some of the dye injection experiments, dye transfer from the injected cell to neighboring cells was observed. Freeze fracture of the bag cell clusters and their surrounding connective tissue revealed numerous gap junctions on bag cell processes within the clusters as well as on more distal processes. We have also examined the morphology and coupling between bag cells in primary culture. As in the intact ganglion, bag cells in culture were found to be multipolar. All pairs of bag cells whose somata or processes had formed contacts in culture were electrically coupled. The strongest coupling was observed between pairs of cells whose somata appeared closely apposed. In these cases transfer of Lucifer Yellow between cells could also be observed. It is therefore likely that the synchrony of bag cell action potentials during a bag cell afterdischarge is a result of coupling between individual cells in the bag cell cluster.

Ductin is a highly conserved and polytopic transmembrane protein which is the subunit c component of the vacuolar H(+)‐ATPase (V‐ATPase) and a component of a connexon channel of gap junctions. Previous studies have suggested that ductin in the V‐ATPase has the opposite orientation of ductin in a connexon. Using an in vitro translation system coupled to microsomes derived from the endoplasmic reticulum, we show that ductin is co‐translationally inserted into the membrane bilayer, suggesting a dependency on the signal recognition particle for synthesis. By attaching a C‐terminal polypeptide derived from beta‐lactamase and by using cysteine replacement coupled to chemical labelling, we show that ductin is inserted into the microsomal membrane in both orientations in similar proportions. In contrast, squid rhodopsin appears to be inserted in a single orientation. Changing conserved charged residues at the N‐terminus of ductin does not affect the ratio of the two orientations. Once in the microsomal membrane, ductin assembles into an oligomeric complex which contains a pore accessible to a water‐soluble probe, reminiscent of the ductin complex found in the V‐ATPase and a connexon.

Human papillomavirus type 16 E5 protein (HPV16 E5) upregulates ligand-mediated activation of the epidermal growth factor receptor (EGFR) in transfected human keratinocytes. HPV16 E5 binds to the 16 kDa proteolipid (subunit c) of the vacuolar H + -ATPase (16K), responsible for endosomal acidi®cation, and this binding has been suggested to be responsible for increased recycling of the EGFRs. Using mutant deletions we show here that amino acids 54 ± 78, but not 79 ± 83 are necessary for binding to the 16K proteolipid. EGF treatment of cells expressing wild type or mutants of the E5 protein show that deletion of the last carboxy terminal 5 amino acids results in loss of E5-mediated EGFR overactivation. Thus, our results show that the binding capacity of HPV16 E5 to 16K can be dissociated from the eect of the viral protein on EGFR activation.