Topology mapping using the HA/Suc2/His4C reporter. (A) Schematic diagram of the membrane protein (MP) reporter fusions. Consensus acceptor sites for N-linked glycosylation are indicated (Y). (B) Growth and N-glycosylation assays. Plasmid-transformed his4⁻ cells were streaked on His-negative plates supplemented with 6 mM histidinol and incubated at 30°C for 2–3 days. N-glycosylation was assessed by Endo H treatment of whole-cell lysates, and identification of MP–reporter fusions was determined by Western blotting using an anti-HA antibody. The Ymr155wp fusion grows on His-negative plates, and the SUC2 domain is not glycosylated, demonstrating that the C terminus is cytosolic (C_in); the Ynr030wp fusion behaves in the opposite way and is, therefore, assigned as C_out.

The yeast membrane proteome. (A) GO annotations (GO slim terms) (35) for the 546 proteins with an assigned C-terminal location. (B) The different GO categories in A subdivided with respect to topology. C_in topologies are plotted upward and C_out downward. Although all predicted single-spanning membrane proteins were excluded from the study, a couple of proteins initially predicted to have two transmembrane helices were predicted with only one transmembrane helix in the C-terminally constrained models. The corresponding results for 601 E. coli inner membrane proteins (15) (scaled to match the 546 yeast proteins) are shown as white bars inside the bars representing the S. cerevisiae proteins.

The C terminus of Ygl263wp is on the extracytosolic side of the membrane. (A) Topology model for Ygl263wp based on a COS family consensus prediction with four transmembrane segments and the experimentally determined C_out orientation. The three potential N-linked glycosylation acceptor sites are indicated (Y). (B) The modification of the three internal N-glycosylation sites in Ygl263wp assessed by Endo H digestion of an HA/His₈-tagged full-length protein lacking the C-terminal topology reporter. Samples were analyzed by 10% SDS/PAGE, followed by Western blotting using an anti-HA antibody. Molecular mass standards are included in lane 3.

Homologs in 38 fully sequenced eukaryotic genomes to 534 assigned S. cerevisiae proteins and to 612 previously analyzed E. coli proteins (15) for which the blast alignment extends to within 15 residues of the C terminus of both query and target proteins. Dark red bars represent the total number of primary and secondary homologs (see Results) that are assigned only by S. cerevisiae proteins in each organism; yellow bars represent primary and secondary homologs assigned only by E. coli proteins; orange bars represent primary and secondary homologs that are assigned by both S. cerevisiae and E. coli proteins. Dark blue bars show the number of unique S. cerevisiae proteins that have at least one homolog in the other genome. Abbreviations are from the Superfamily database (38): hs, H. sapiens; xp, P. troglodytes; mm, M. musculus; rn, R. norvegicus; gg, G. gallus; da, D. rerio; to, F. rubripes; dm, D. melanogaster; ag, A. gambiae; xe, X. tropicalis; ci, C. intestinalis; cw, C. briggsae; ce, C. elegans; dt, D. discoideum; at, A. thaliana; oj, O. sativa ssp. japonica; os, O. sativa ssp. indica; pl, P. falciparum; py, P. yoelii ssp. yoelii; eu, E. cuniculi; tb, T. brucei; um, U. maydis; po, S. pombe; ns, N. crassa; gr, M. grisea; go, A. gossypii; na, A. nidulans; fg, F. graminearum; al, C. albicans; yl, Y. lipolytica; dh, D. hansenii; gl, C. glabrata; kl, K. lactis; kw, K. waltii; y1, S. bayanus; y6, S. mikatae; y8, S. paradoxus; sc, S. cerevisiae.