Assessment of Topogenic Functions of Anticipated Transmembrane Segments of Human Band 3

Abstract

Band 3 protein is a typical multispanning membrane protein whose membrane topology has been extensively studied from various protein chemical approaches. To clarify the membrane topogenesis of this multispanning protein on the endoplasmic reticulum, the topogenic functions of the anticipated transmembrane segments were individually assessed in an in vitro system using two series of model proteins in which each segment was placed in either a "stop-transfer" context or a "translocation initiation" context. They were expressed in a cellfree system containing rough microsomal membranes, and their topologies were evaluated by taking advantage of either sensitivity to protease or accessibility to N-glycosylation. We found that some segments seem to possess insufficient topogenic functions for membrane integration: the second transmembrane segment (TM2) is insufficient for the stop-transfer sequence, and TM3, TM5, and TM7 are not sufficient for the translocation initiation. In contrast to these phenomena, we herein demonstrate that TM2 shows an efficient stop-transfer function when it is near the preceding TM1 and suggest that TM3, TM5, and TM7 are followed by TM segments with a strong topogenic function to form N exo /C cyt topology, via which the preceding segments are integrated into the membrane. From these results, we propose that the interactions between the TMs should be operative during membrane integration, and that the segments with a weak topogenic function are given a transmembrane orientation by their following TMs.

Structural model for the organization of the transmembrane spans of the human red-cell anion exchanger (band 3; AE1)

“…Taken together, these results suggest that span 5 might contribute (either directly or indirectly) to the interface between the fragment and b3mem, and that span 6 might assist. It has been suggested recently [25,26] that span 6 might assist in the correct TM integration of span 5.…”

Topogenesis of Two Transmembrane Type K+ Channels, Kir 2.1 and KcsA

“…Y. Kubo and L. Y. Jan (20) and Dr. R. MacKinnon (9). To assess the topogenic function of the individual segments, each PCRamplified fragment was subcloned into the corresponding sites in the pCITE-2a-based plasmids used for topogenic assay (21). Fig.…”

“…2, 3, and 5. The cDNAs encoding wild-type prolactin (PL), PL containing an N-glycosylation site, or the type I signal-anchor sequence (SA-I) transmembrane region of the Escherichia coli leader peptidase (H1) were derived from plasmids described previously (21). In some constructs, the Kir 2.1 Trp 81 -Phe 103 coding sequence was replaced with the coding sequence for Met 1 -Leu 28 from mouse dipeptidyl peptidase IV (DPP IV), which is an SA-II transmembrane segment.…”

“…All site-directed mutations were carried out using a two-step PCR approach (22). The RNAs were translated in the reticulocyte lysate system (21,23) in the absence or presence of dog pancreas rough microsomal membranes (RM) (21,24). Proteinase K was used for protease treatment; in all protease experiments, RM was checked for lack of leakage, which would affect the protease results, by testing the effect of addition of 0.5% Triton X-100 on the observed results.…”

Requirement of Negative Residues, Asp 95 and Asp 105, in S2 on Membrane Integration of a Voltage-dependent K+Channel, KAT1

“…[5][6][7] The RNAs were translated in a reticulocyte lysate 7,8) in either the absence or presence of RM. 7,9) This basic unit was then modiˆed by replacement of D95 and W or D105 with serine, S. If S3 and S4 are correctly integrated, the glycosylation site lies within the RM lumen and is glycosylated, whereas PLgly is not glycosylated. Integration of S3 and S4 in the presence of RM can therefore be detected by mono-glycosylation of the PLgly construct.…”