Fanarraga M L, Párraga M, Aloria K, del Mazo J, Avila J, Zabala J C
Departamento de Biología Molecular, Universidad de Cantabria, Santander, Spain.
Cell Motil Cytoskeleton. 1999;43(3):243-54. doi: 10.1002/(SICI)1097-0169(1999)43:3<243::AID-CM7>3.0.CO;2-0.
The correct folding of tubulins and the generation of functional alpha beta-tubulin heterodimers require the participation of a series of recently described molecular chaperones and CCT (or TRiC), the cytosolic chaperonin containing TCP-1. p14 (cofactor A) is a highly conserved protein that forms stable complexes with beta-tubulin which are not apparently indispensable along the in vitro beta-tubulin folding route. Consequently, the precise role of p14 is still unknown, though findings on Rb12p (its yeast homologue) suggest p14 might play a role in meiosis and/or perhaps to serve as an excess beta-tubulin reservoir in the cell. This paper investigates the in vivo possible role of p14 in testis where mitosis, meiosis, and intense microtubular remodeling processes occur. Our results confirm that p14 is more abundantly expressed in testis than in other adult mammalian tissues. Northern blot, Western blot, in situ hybridization, and immunocytochemical analyses have all demonstrated that p14 is progressively upregulated from the onset of meiosis through spermiogenesis, being more abundant in differentiating spermatids. The close correlation observed between the mRNA expression waves for p14 and testis specific tubulin isotypes beta 3 and alpha 3/7, together with the above results, suggest that p14 role in testis would presumably be associated to beta-tubulin processing rather than meiosis itself. Additional in vitro beta 3-tubulin synthesis experiments have shown that p14 plays a double role in beta-tubulin folding, enhancing the dimerization of newly synthesized beta-tubulin isotypes as well as capturing excess beta-tubulin monomers. The above evidence suggests that p14 is a chaperone required for the actual beta-tubulin folding process in vivo and storage of excess beta-tubulin in situations, such as in testis, where excessive microtubule remodeling could lead to a disruption of the alpha-beta balance. As seen for other chaperones, p14 could also serve as a route to lead excess beta-tubulin or replaced isotypes towards degradation.
微管蛋白的正确折叠以及功能性αβ-微管蛋白异二聚体的生成需要一系列最近描述的分子伴侣和CCT（或TRiC，即含TCP-1的胞质伴侣蛋白）的参与。p14（辅因子A）是一种高度保守的蛋白质，它与β-微管蛋白形成稳定的复合物，在体外β-微管蛋白折叠途径中这些复合物显然并非不可或缺。因此，p14的确切作用仍然未知，不过关于Rb12p（其酵母同源物）的研究结果表明，p14可能在减数分裂中发挥作用，和/或可能在细胞中作为过量β-微管蛋白的储存库。本文研究了p14在睾丸中的体内可能作用，睾丸中会发生有丝分裂、减数分裂以及强烈的微管重塑过程。我们的结果证实，p14在睾丸中的表达比在其他成年哺乳动物组织中更为丰富。Northern印迹、Western印迹、原位杂交和免疫细胞化学分析均表明，从减数分裂开始到精子发生过程中，p14表达逐渐上调，在分化的精子细胞中更为丰富。p14的mRNA表达波动与睾丸特异性微管蛋白亚型β3和α3/7之间观察到的密切相关性，以及上述结果表明，p14在睾丸中的作用可能与β-微管蛋白的加工有关，而非与减数分裂本身相关。额外的体外β3-微管蛋白合成实验表明，p14在β-微管蛋白折叠中发挥双重作用，既增强新合成的β-微管蛋白亚型的二聚化，又捕获过量的β-微管蛋白单体。上述证据表明，p14是体内实际β-微管蛋白折叠过程以及在诸如睾丸这种过度微管重塑可能导致α-β平衡破坏的情况下储存过量β-微管蛋白所必需的伴侣蛋白。正如其他伴侣蛋白一样，p14也可能作为一条途径，引导过量的β-微管蛋白或被取代的亚型走向降解。
