Volume 2, Issue 4, Pages 495-503 (October 1998) Forced Transmembrane Orientation of Hydrophilic Polypeptide Segments in Multispanning Membrane Proteins  Kazuhisa Ota, Masao Sakaguchi, Gunnar von Heijne, Naotaka Hamasaki, Katsuyoshi Mihara  Molecular Cell  Volume 2, Issue 4, Pages 495-503 (October 1998) DOI: 10.1016/S1097-2765(00)80149-5

Figure 1 TM8 Can Translocate the L7 Loop in the Absence of TM7 (A) Constructs used for assessing the function of TM8 in the translocation of the L7 loop. A segment encoding TM1 to TM8 of band 3 was fused with mature prolactin (1-8-PL). TM7 of 1-8-PL was replaced with a hydrophilic sequence (Thr-31 to Phe-80) from prolactin (1-8Δ7-PL). TM8 was deleted from this construct (1-6-PL). The L7 loop and TM8 were fused to mature prolactin (G8-PL). The hydrophilic sequence replacing TM7 and the N-glycosylation site are indicated by a closed box and an open circle, respectively. The reporter domain of mature prolactin in the carboxy-terminal portion is indicated by an open box (PL). (B) Constructs were expressed in a cell-free system in the presence of RM. After the translation reaction, membranes were precipitated by centrifugation through a cushion containing 0.5 M KCl and analyzed by SDS-PAGE. Aliquots of the membrane precipitates were treated by endoglycosidase H (EndoH +). Translocation of the glycosylated loop was monitored by its N-glycosylation. The N-glycosylation efficiency (glc) is indicated. (C) Possible membrane topologies. Closed boxes indicate the hydrophilic sequence replacing TM7, and open boxes indicate the TM segments of band 3. (D) Hydrophobicity plots of the TM1-8 region of wild-type and TM7-exchanged constructs. Each sequence was analyzed by TopPred II using the default parameters (Claros and von Heijne 1994). The line at <H> = 1 indicates the standard cutoff value for predicting “certain” TM segments. Position 1 corresponds to Gly-376 of the band 3 protein, and the TMs are indicated by boxed numbers. Molecular Cell 1998 2, 495-503DOI: (10.1016/S1097-2765(00)80149-5)

Figure 2 Forced Transmembrane Orientation of the Hydrophilic Segment in Model Proteins with Two Hydrophobic Nexo/Ccyt Segments (A) The H1 segment of E. coli leader peptidase and TM8 from band 3 were positioned as in the figure. N-glycosylation sites are shown by open circles. H1 and TM8 were separated by 100 or 200 hydrophilic residues from prolactin. (B) Constructs were expressed in vitro. After translation in the presence of RM (+), aliquots were treated by the proteinase K and/or EndoH. Proteinase K–resistant fragments are indicated by arrowheads. Aliquots were extracted under the alkali conditions and separated into membrane-bound (M) and soluble (S) fractions. (C) Possible topologies. Potential N-glycosylation sites accessible to the oligosaccharyl transferase in the lumen are indicated by open circles. (D) Transmembrane orientation of a hydrophilic segment induced by the SA-I of synaptotagmin 2. The N-terminal tail and the SA-I of synaptotagmin 2 were introduced instead of TM8 of band 3. N-glycosylation sites (indicated by circles) were positioned 17 residues before H1 and 29 residues before the synaptotagmin 2 transmembrane segment. Constructs were expressed in vitro in the absence (−) or presence (+) of RM. Aliquots were treated by proteinase K or EndoH. The diglycosylated form is indicated by two dots. Molecular Cell 1998 2, 495-503DOI: (10.1016/S1097-2765(00)80149-5)

Figure 3 N-glycosylation Efficiency Depends on the Distance from H1 (A) Constructs used for glycosylation site scanning. N-glycosylation consensus sites were created in the indicated position (X residues from H1 segment) of H1-100-8 construct. (B) Constructs were expressed in vitro in the presence (+) or absence (−) of RM. (C) Quantitation of the data in (B). Molecular Cell 1998 2, 495-503DOI: (10.1016/S1097-2765(00)80149-5)

Figure 4 Membrane Topology Assessed by Factor Xa Cleavage (A) Factor Xa sites (Ile-Glu-Gly-Arg) were created 12 residues after H1 (H1-Xa-N) or 10 residues before TM8 (H1-Xa-C) in the H1-100-8 construct. (B) After synthesis in vitro in the presence of RM, membranes were isolated and treated with factor Xa (8 μg/ml) and analyzed by SDS-PAGE. The di- and monoglycosylated forms are indicated by double and single dots, respectively. Proteolytic fragments are indicated by arrowheads. (C) Relative contents of mono- and diglycosylated forms before and after factor Xa cleavage. Molecular Cell 1998 2, 495-503DOI: (10.1016/S1097-2765(00)80149-5)

Figure 5 TM3 Is Integrated by the SA-I Function of TM4 (A) Constructs used to assess the SA-I function of TM4. The region from TM1 to TM4 was fused to mature prolactin, and the L7 loop was introduced between the TM3 and TM4 segments (1-G4-PL). The N-glycosylation site was placed 3 or 13 residues from TM3 (3G and 13G, respectively). TM4 was deleted from 1-G4-PL (1-3G-PL). (B) Constructs were expressed in vitro in the absence (−) or presence (+) of RM. Aliquots were treated EndoH or proteinase K. (C) Topology of the constructs in the membrane. Molecular Cell 1998 2, 495-503DOI: (10.1016/S1097-2765(00)80149-5)

Figure 6 H1-100-8 Does Not Jam the Translocon (A) Effect of prior insertion of membrane proteins on the translocation of preprolactin. mRNAs encoding synaptoagmin (Stg; lanes 2 and 6), full-length H1-100-8 (full; lanes 3 and 7), and truncated H1-100-8 (trun; lanes 4 and 8) were translated in the presence of limiting amounts of RM at 30°C for 20 min. As a mock reaction, the first translation was carried out without added mRNA (lanes 1 and 5). After the translation with the first mRNA, the reactions were diluted 4-fold with newly prepared translation mixture including preprolactin mRNA, and the mixtures were further incubated at 22°C for 90 min. Processed, mature prolactin is indicated by an arrow, and the di- and monoglycosylated products are indicated by double and single dots. (B) Relative processing efficiency of the secondary synthesized preprolactin. The processing efficiency is expressed as a ratio (%) to that obtained after the first mock reaction (lanes 1 and 5). The results shown in the figure represent the average of three experiments with standard deviations (error bars). The relative amounts of the glycosylated forms of Stg2, full-length H1-100-8, and truncated H1-100-8 in lanes 6–8 are 1.2, 1.9, and 1.0, respectively. Molecular Cell 1998 2, 495-503DOI: (10.1016/S1097-2765(00)80149-5)

Figure 7 Models for Cotranslational Integration of Multispanning Membrane Proteins (A) A transmembrane segment not sufficiently hydrophobic to open the translocation channel by itself (a–b) is pulled across the translocon by the following Nexo/Ccyt SA-I (c–d). (B) A sufficiently hydrophobic Ncyt/Cexo SA-II segment opens the translocation channel and promotes translocation of the following portion of the chain (f–g). The next transmembrane segment stops translocation (St function, h). Molecular Cell 1998 2, 495-503DOI: (10.1016/S1097-2765(00)80149-5)