Retrograde transport from the pre-Golgi intermediate compartment and the Golgi complex is affected by the vacuolar H+-ATPase inhibitor bafilomycin A1.

The effect of the vacuolar H+-ATPase inhibitor bafilomycin A1 (Baf A1) on the localization of pre-Golgi intermediate compartment (IC) and Golgi marker proteins was used to study the role of acidification in the function of early secretory compartments. Baf A1 inhibited both brefeldin A- and nocodazole-induced retrograde transport of Golgi proteins to the endoplasmic reticulum (ER), whereas anterograde ER-to-Golgi transport remained largely unaffected. Furthermore, p58/ERGIC-53, which normally cycles between the ER, IC, and cis-Golgi, was arrested in pre-Golgi tubules and vacuoles, and the number of p58-positive approximately 80-nm Golgi (coatomer protein I) vesicles was reduced, suggesting that the drug inhibits the retrieval of the protein from post-ER compartments. In parallel, redistribution of beta-coatomer protein from the Golgi to peripheral pre-Golgi structures took place. The small GTPase rab1p was detected in short pre-Golgi tubules in control cells and was efficiently recruited to the tubules accumulating in the presence of Baf A1. In contrast, these tubules showed no enrichment of newly synthesized, anterogradely transported proteins, indicating that they participate in retrograde transport. These results suggest that the pre-Golgi structures contain an active H+-ATPase that regulates retrograde transport at the ER-Golgi boundary. Interestingly, although Baf A1 had distinct effects on peripheral pre-Golgi structures, only more central, p58-containing elements accumulated detectable amounts of 3-(2, 4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP), a marker for acidic compartments, raising the possibility that the lumenal pH of the pre-Golgi structures gradually changes in parallel with their translocation to the Golgi region.

Endocytic pathway from the basal plasma membrane to the ruffled border membrane in bone-resorbing osteoclasts.

We have characterized the convoluted ruffled border (RB) membrane that an activated osteoclast maintains against the bone matrix. The bulk of both lgp110 and rab7, a small GTP-binding protein participating in vesicle fusion to late endosomes, was localized to the RB. This indicates that the membrane has some characteristics of late endosomal membranes in other cells. Furthermore, the bulk of membrane-bound rab7 on the RB suggests that endocytic membrane transport is oriented towards the RB in resorbing osteoclasts. Consistently, both lumenal horseradish peroxidase and receptor-bound transferrin, a marker of the early endosomal recycling pathway, were efficiently endocytosed from the basal plasma membrane and delivered to the RB. Delivery of membrane-associated transferrin to the RB further indicates that the RB is compositionally different from lysosomes and suggests that the endocytic pathway contributes to the maintenance of functional RB. In addition to transporting receptor-bound cargo to the RB, the endocytic pathway could act in balancing the membrane traffic associated with transcytosis from the RB to the basal plasma membrane. Endocytic processes (retrieval of mannose 6-phosphate receptors) in osteoclasts appeared to be fairly sensitive to bafilomycin A1, a specific inhibitor of vacuolar-type proton ATPases. Thus blocking the endocytic membrane traffic towards the RB could explain the inactivation of cells by low concentrations of the drug.

Characterization and cellular distribution of the osteoclast ruffled membrane vacuolar H+-ATPase B-subunit using isoform-specific antibodies.

Acidification of the bone surface, leading to bone resorption, is accomplished by a vacuolar-type H+-ATPase present in a specialized domain of the plasma membrane of the osteoclast known as the ruffled membrane. Structure and function appears to be highly conserved within this class of multisubunit enzymes. However, cloning and sequencing of complementary DNA has shown that one of the subunits in the catalytic domain, the B-subunit, exists in at least two forms, B1 and B2. B1 messenger RNA has been found almost exclusively in the kidney, whereas messenger RNA for B2 has been found in all tissues studied, including the kidney. It has been speculated that the B1 isoform might be involved in targeting to the plasma membrane. In the present study, we have characterized the B-subunit of the chicken osteoclast H+-ATPase using antibodies directed against peptides with isoform-specific or conserved sequences of the B-subunit. Western analysis was performed on chicken osteoclast membrane vesicles and on partially purified chicken osteoclast H+-ATPase and was compared with similar analysis of H+-ATPase isolated from bovine kidney and brain. The B1-specific antibody reacted with a polypeptide of approximately 56 kD on immunoblots of the renal H+-ATPase, whereas no reaction could be detected against the osteoclast H+-ATPase or the osteoclast membrane vesicle preparation. In contrast, the antibody against a B2-specific sequence reacted with a peptide of approximately 56 kD on immunoblots of the osteoclast H+-ATPase, the renal H+-ATPase, and the clathrin-coated vesicle H+-ATPase. The antibody against a conserved region of the B-subunit did not generate any evidence for the presence of isoforms other than B2 in the osteoclast. Immunocytochemistry of rat osteoclasts on bovine bone slices using the B2 antibody showed intense polarized staining along the plasma membrane facing the bone surface in actively resorbing osteoclasts whereas nonresorbing osteoclasts were diffusely stained throughout the cytoplasm. By confocal microscopy, the B2 staining was located to the level of the ruffled membrane and appeared to be concentrated to the peripheral areas of the membrane adjacent to the sealing zone. We conclude that the osteoclast vacuolar H+-ATPase contains the B2 isoform and suggest that upon initiation of resorption the pump is translocated from the cell interior to a special domain of the ruffled membrane close to the sealing zone.

Bone-resorbing osteoclasts reveal a dynamic division of basal plasma membrane into two different domains.

Bone-resorbing multinucleate osteoclasts exhibit a ruffled border membrane apposing the bone and a basal membrane contacting the circulation. A junctional complex called the sealing zone separates these two membrane domains, but the defined nature of these membrane domains has remained obscure. We now show, using enveloped viral glycoproteins and lectins as tools, that osteoclasts exhibit a novel membrane domain in the basal surface when they are polarized for resorption. Influenza haemagglutinin, which is apically targeted in epithelial cells, is targeted to a restricted area at the top of the basal surface, while vesicular stomatitis virus G-protein which is basolaterally targeted in epithelia, occupies the rest of the basal surface. Neither of these viral glycoproteins is gathered to the ruffled border nor sealing zone area, but they share in a specific way the basal surface. To show that the division of basal membrane into two different domains also occurs in non-infected cells, we have analyzed the distribution of receptors for these viruses and binding sites of some lectins. Both of these methods show that also some endogenous proteins are located in different domains in the basal surface in active osteoclasts. We also show that these two different membrane domains can be distinguished in scanning electron microscopy level due to the villus appearance of the central basal domain.

Active vacuolar H+ATPase is required for both endocytic and exocytic processes during viral infection of BHK-21 cells.

Bafilomycin A1 (Baf), a specific inhibitor of the vacuolar-type proton pump, inhibited the entry of Semliki Forest virus and vesicular stomatitis virus into BHK-21 cells. The inhibition occurred at concentrations that dissipated intracellular acidic compartments. Viral infection was totally inhibited by 30 nM Baf while endocytosis of the virus or fluorescein isothiocyanate-dextran was not affected. Thus, a vacuolar-type proton pump was responsible for acidification of the endosomes needed for virus entry. When the cells were exposed to 100 nM Baf after virus entry, viral glycoprotein synthesis continued normally. The viral glycoproteins acquired resistance to endoglycosidase H, indicating arrival in the medial Golgi apparatus. However, maturation processes occurring in the trans-Golgi compartment were inhibited, and the amounts of viral glycoproteins appearing at the cell surface were reduced. Double immunofluorescence studies showed that in the presence of Baf the viral glycoproteins were found in and around mannosidase II-positive Golgi structures. To analyze whether Baf blocked transport from the trans-Golgi compartment to the cell surface, the viral glycoproteins were allowed to accumulate in the trans-Golgi network by utilizing a 20 degrees C block. Subsequent incubation at 37 degrees C in the presence of Baf did not inhibit the terminal maturation processes or transport to the cell surface, suggesting that the block was before the trans-Golgi network. These results indicate that an active vacuolar-type proton pump in the Golgi apparatus is essential for protein transport in BHK-21 cells.

Age-related changes in the concentration of hydroxypyridinium crosslinks in functionally different skeletal muscles.

High-performance liquid chromatography methods were developed to measure the concentration of hydroxypyridinium crosslinks in the intramuscular collagen and tendinous parts of functionally different skeletal muscles at different ages. A significant increase in pyridinoline concentration took place during maturation reaching 0.32 +/- 0.07 (mol/mol collagen) in soleus (slow plantar flexor) and 0.28 +/- 0.07 in plantaris (fast "mixed" plantar flexor) at the age of 4 months. In medial and lateral gastrocnemius (fast "mixed" plantar flexors) the pyridinoline concentrations (mol/mol collagen) reached 0.24 +/- 0.06 and 0.19 +/- 0.04, respectively, similar to those in both the extensor digitorum longus (fast "mixed" dorsi flexor) and rectus femoris (fast "mixed" knee extensor) muscles, but higher than in the fast "mixed" dorsi flexor muscle, anterior tibialis (0.11 +/- 0.05 mol/mol). By comparison, pyridinoline concentrations of 0.33 mol/mol collagen (+/- 0.10) was measured from longissimus dorsi, a slow-twitch back posture muscle. After maturation the most significant increase in pyridinoline concentration was measured in soleus and gastrocnemius muscles. No differences in the crosslinking between different parts of muscle belly were noticed at any time-point. However, significantly fewer pyridinoline crosslinks were found in tendinous parts of soleus, extensor digitorum longus and anterior tibialis than in intramuscular collagen. The concentration of pyridinoline crosslinks tended to be highest in slow-twitch postural muscles, soleus and longissimus dorsi, and generally higher in plantar flexors which are exposed to higher stretch than dorsal flexors. The reasons for the unexpectedly low concentrations of pyridinoline crosslinks in the tendinous parts of muscles remain to be clarified.