The Saccharomyces cerevisiae vacuolar membrane proton-translocating ATPase (V-ATPase) can be divided into a peripheral membrane complex (V 1 ) containing at least eight polypeptides of 69, 60, 54, 42, 32, 27, 14, and 13 kDa, and an integral membrane complex (V 0 ) containing at least five polypeptides of 100, 36, 23, 17, and 16 kDa. Other yeast genes have been identified that are required for V-ATPase assembly but whose protein products do not co-purify with the enzyme complex. One such gene, VMA12, encodes a 25-kDa protein (Vma12p) that is predicted to contain two membrane-spanning domains. Biochemical analysis has revealed that Vma12p behaves as an integral membrane protein with both the N and C termini oriented toward the cytosol, and this protein immunolocalizes to the endoplasmic reticulum (ER). In cells lacking Vma12p (vma12⌬), the 100-kDa subunit of the V 0 complex (which contains six to eight putative membrane-spanning domains) was rapidly degraded (t1 ⁄2 ϳ 30 min). Protease protection assays revealed that the 100-kDa subunit was inserted/translocated correctly into the ER membrane of vma12⌬ cells. These data indicate that Vma12p functions in the ER after the insertion of V 0 subunits into the ER membrane. We propose that Vma12p functions directly in the assembly of the V 0 subunits into a complex in the ER, and that assembly is required for the stability of the V 0 subunits and their transport as a complex out of this compartment.The yeast vacuolar proton-translocating ATPase (VATPase) 1 belongs to a family of multisubunit vacuolar-type ATPases found in all eukaryotic cells (1, 2). V-ATPases acidify the lumen of many organelles, including vacuoles, lysosomes, endosomes, the Golgi apparatus, as well as clathrin-coated and secretory vesicles (2). V-ATPases play a role in many cellular processes, such as zymogen activation, receptor-mediated endocytosis, and protein sorting (3). The proton gradient generated by the yeast V-ATPase drives the transport of ions, basic amino acids, metabolites, and storage sugars into the vacuole (4 -6).The yeast V-ATPase is similar in both structure and subunit composition to the F 1 F 0 -ATP synthase of mitochondria. The V-ATPase can be divided into a peripherally associated complex (V 1 ) containing ATP hydrolytic and regulatory domains and an integral membrane complex (V 0 ) housing the proton pore (7-8). Biochemical and genetic analyses have demonstrated that the yeast V-ATPase contains at least 13 subunits. V 1 subunits that have been identified include hydrophilic polypeptides of 69, 60, 54, 42, 32, 27, 14, and 13 kDa (2, 9). The V 0 complex includes integral membrane proteins of 100, 23, 17, and 16 kDa and a hydrophilic protein of 36 kDa (10 -12). Yeast cells contain two isoforms of the 100-kDa V 0 subunit, encoded by the VPH1 and STV1 genes (Vph1p, Ref. 10; Stv1p, Ref. 13).Yeast cells lacking any one of the VMA genes exhibit a common set of phenotypes, including pH-sensitive growth and a loss of V-ATPase activity (14 -17). Cells lacking a V 1 subunit fail to assemble ...

We describe a laboratory exercise in DNA profiling that can be used in an undergraduate biochemistry teaching laboratory to demonstrate four fundamental procedures: isolation of genomic DNA from human (cheek) cells, use of the polymerase chain reaction (PCR) to amplify DNA, separation of amplified DNAs on agarose and polyacrylamide gels, and quantitative analysis of data (while comparing two different gel separation techniques). The target of PCR amplification is the 369–801 base pair D1S80 locus of the human genome, a variable number of tandem repeats (VNTR) locus containing a 16 base pair repeat unit. Students amplify DNA from their entire class, determine the size and genotype of all of the students' D1S80 loci by two gel separation techniques, compare the samples to an "evidence" sample to solve a fictitious crime, and critically evaluate the methodology.