Schapiro F B, Grinstein S
Cell Biology Programme, Research Institute, The Hospital for Sick Children and the Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1X8, Canada.
J Biol Chem. 2000 Jul 14;275(28):21025-32. doi: 10.1074/jbc.M002386200.
The factors contributing to the establishment of the steady state Golgi pH (pH(G)) were studied in intact and permeabilized mammalian cells by fluorescence ratio imaging. Retrograde transport of the nontoxic B subunit of verotoxin 1 was used to deliver pH-sensitive probes to the Golgi complex. To evaluate whether counter-ion permeability limited the activity of the electrogenic V-ATPase, we determined the concentration of K(+) in the lumen of the Golgi using a null point titration method. The [K(+)] inside the Golgi was found to be close to that of the cytosol, and increasing its permeability had no effect on pH(G). Moreover, the capacity of the endogenous counter-ion permeability exceeded the rate of H(+) pumping, implying that the potential across the Golgi membrane is negligible and has little influence on pH(G). The V-ATPase does not reach thermodynamic equilibrium nor does it seem to be allosterically inactivated at the steady state pH(G). In fact, active H(+) pumping was detectable even below the resting pH(G). A steady state pH was attained when the rate of pumping was matched by the passive backflux of H(+) (equivalents) or "leak." The nature of this leak pathway was investigated in detail. Neither vesicular traffic nor H(+)/cation antiporters or symporters were found to contribute to the net loss of H(+) from the Golgi. Instead, the leak was sensitive to voltage changes and was inhibited by Zn(2+), resembling the H(+) conductive pathway of the plasma membrane. We conclude that a balance between an endogenous leak, which includes a conductive component, and the H(+) pump determines the pH at which the Golgi lumen attains a steady state.
通过荧光比率成像技术，在完整的和通透的哺乳动物细胞中研究了有助于建立稳态高尔基体pH值（pH(G)）的因素。利用1型志贺毒素的无毒B亚基的逆行转运将pH敏感探针递送至高尔基体复合体。为了评估抗衡离子通透性是否限制了电生性V-ATP酶的活性，我们使用零点滴定法测定了高尔基体腔中K(+)的浓度。发现高尔基体内部的[K(+)]与细胞质中的接近，增加其通透性对pH(G)没有影响。此外，内源性抗衡离子通透性的能力超过了H(+)泵出的速率，这意味着高尔基体膜两侧的电位可以忽略不计，对pH(G)影响很小。V-ATP酶在稳态pH(G)时未达到热力学平衡，似乎也没有变构失活。事实上，即使在静息pH(G)以下也能检测到活跃的H(+)泵出。当泵出速率与H(+)（当量）的被动回流或“渗漏”相匹配时，达到了稳态pH。详细研究了这种渗漏途径的性质。未发现囊泡运输、H(+)/阳离子反向转运体或同向转运体对高尔基体中H(+)的净损失有贡献。相反，这种渗漏对电压变化敏感，并受到Zn(2+)的抑制，类似于质膜的H(+)传导途径。我们得出结论，内源性渗漏（包括一个传导成分）和H(+)泵之间的平衡决定了高尔基体腔达到稳态时的pH值。
