Gray L, Baker K, Kenny B, Mackman N, Haigh R, Holland I B
Department of Genetics, University of Leicester, UK.
J Cell Sci Suppl. 1989;11:45-57. doi: 10.1242/jcs.1989.supplement_11.4.
Escherichia coli haemolysin (HlyA), a 107K (K = 10(3) Mr) protein, is secreted to the medium in an hlyB, hlyD-dependent process. Secretion, however, depends on neither an N-terminal signal sequence nor on SecA, which is part of the normal cellular export machinery for periplasmic and outer membrane proteins. In contrast, HlyA contains a novel C-terminal secretion signal encompassing the last 27 amino acids and possibly some additional residues immediately upstream. This region is characterized by a 16 residue 'aspartic acid box' composed largely of small amino acids which we propose constitutes an important element in recognition of the membrane translocation complex constituted by HlyB and HlyD. This feature is also found at the C-terminus of the adenyl cyclase and leukotoxin A molecules and resembles a recently identified eukaryotic C-terminal signal for targeting to glycosomes. A domain of the HlyB component of the haemolysin transport system is also similar to a domain widely distributed in nature, apparently acting as an ATP-dependent transport protein for a wide variety of molecules. Secretion of haemolysin, however, is the first example of a protein translocation system involving an HlyB-like molecule. This suggests that a major role of HlyB or at least its C-terminal domain is the coupling of energy to translocation of the haemolysin. It is more likely therefore that HlyD is more involved in the actual translocation through the membrane. On the basis of genetical and biochemical studies we propose that the haemolysin is translocated directly to the medium bypassing the periplasm. We further propose that HlyB and HlyD together constitute a membrane-bound translocator specific for molecules bearing the HlyA targeting sequence, and that the organization of this complex (conceivably involving other E. coli membrane proteins) must somehow straddle the inner and outer membranes. Finally, the HlyA C-terminal domain has been successfully used to promote the secretion to the medium of a number of heterologous polypeptides, in an HlyB,D-dependent manner.
大肠杆菌溶血素（HlyA）是一种107K（K = 10³ 道尔顿分子量）的蛋白质，通过hlyB、hlyD依赖的过程分泌到培养基中。然而，分泌既不依赖于N端信号序列，也不依赖于SecA，SecA是用于周质和外膜蛋白的正常细胞输出机制的一部分。相反，HlyA包含一个新的C端分泌信号，包括最后27个氨基酸以及可能紧接其上游的一些额外残基。该区域的特征是一个由16个残基组成的“天冬氨酸盒”，主要由小氨基酸组成，我们认为它是识别由HlyB和HlyD构成的膜转运复合物的重要元件。在腺苷酸环化酶和白细胞毒素A分子的C端也发现了这一特征，并且类似于最近鉴定出的用于靶向糖体的真核生物C端信号。溶血素转运系统的HlyB组分的一个结构域也类似于自然界中广泛分布的一个结构域，显然作为多种分子的ATP依赖性转运蛋白起作用。然而，溶血素的分泌是涉及HlyB样分子的蛋白质转运系统的首个例子。这表明HlyB或至少其C端结构域的主要作用是将能量与溶血素的转运相偶联。因此更有可能的是，HlyD更多地参与了实际的跨膜转运。基于遗传学和生化研究，我们提出溶血素绕过周质直接转运到培养基中。我们进一步提出，HlyB和HlyD共同构成一个对带有HlyA靶向序列的分子具有特异性的膜结合转运体，并且这个复合物的组织（可以想象涉及其他大肠杆菌膜蛋白）必须以某种方式跨越内膜和外膜。最后，HlyA的C端结构域已成功用于以HlyB、D依赖的方式促进多种异源多肽分泌到培养基中。
