Despite advances in resolving the structures of multi-pass membrane proteins, little is known about the native folding pathways of these complex structures. Using single-molecule magnetic tweezers, we here report a folding pathway of purified human glucose transporter 3 (GLUT3) reconstituted within synthetic lipid bilayers. The N-terminal major facilitator superfamily (MFS) fold strictly forms first, serving as a structural template for its C-terminal counterpart. We found polar residues comprising the conduit for glucose molecules present major folding challenges. The endoplasmic reticulum membrane protein complex facilitates insertion of these hydrophilic transmembrane helices, thrusting GLUT3’s microstate sampling toward folded structures. Final assembly between the N- and C-terminal MFS folds depends on specific lipids that ease desolvation of the lipid shells surrounding the domain interfaces. Sequence analysis suggests that this asymmetric folding propensity across the N- and C-terminal MFS folds prevails for metazoan sugar porters, revealing evolutionary conflicts between foldability and functionality faced by many multi-pass membrane proteins.

尽管在解析多通道膜蛋白的结构方面取得了进展，但对这些复杂结构的天然折叠途径知之甚少。使用单分子磁性镊子，我们在这里报告了在合成脂质双层内重组的纯化人类葡萄糖转运蛋白 3 (GLUT3) 的折叠途径。N 端主要促进子超家族 (MFS) 严格首先形成折叠，作为其 C 端对应物的结构模板。我们发现构成葡萄糖分子管道的极性残基提出了主要的折叠挑战。内质网膜蛋白复合物有助于插入这些亲水性跨膜螺旋，将 GLUT3 的微观状态采样推向折叠结构。N 端和 C 端 MFS 折叠之间的最终组装取决于特定的脂质，这些脂质可以缓解域界面周围脂质壳的去溶剂化。序列分析表明，这种跨越 N 端和 C 端 MFS 折叠的不对称折叠倾向在后生动物糖搬运工中普遍存在，揭示了许多多通道膜蛋白所面临的可折叠性和功能性之间的进化冲突。