Harasaki Kouki, Lubben Nienke B, Harbour Michael, Taylor Marcus J, Robinson Margaret S
University of Cambridge, Department of Clinical Biochemistry, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK.
Traffic. 2005 Nov;6(11):1014-26. doi: 10.1111/j.1600-0854.2005.00341.x.
The AP-1 and AP-2 complexes are the most abundant adaptors in clathrin-coated vesicles (CCVs), but clathrin-mediated trafficking can still occur in the absence of any detectable AP-1 or AP-2. To find out whether adaptor abundance reflects cargo abundance, we used lectin pulldowns to identify the major membrane glycoproteins in CCVs from human placenta and rat liver. Both preparations contained three prominent high molecular-weight proteins: the cation-independent mannose 6-phosphate receptor (CIMPR), carboxypeptidase D (CPD) and low-density lipoprotein receptor-related protein 1 (LRP1). To investigate how these proteins are sorted, we constructed and stably transfected CD8 chimeras into HeLa cells. CD8-CIMPR localized mainly to early/tubular endosomes, CD8-CPD to the trans Golgi network and CD8-LRP1 to late/multivesicular endosomes. All three constructs redistributed to the plasma membrane when clathrin was depleted by siRNA. CD8-CIMPR was also strongly affected by AP-2 depletion. CD8-CPD was moderately affected by AP-2 depletion but strongly affected by depleting AP-1 and AP-2 together. CD8-LRP1 was only slightly affected by AP-2 depletion; however, mutating an NPXY motif in the LRP1 tail caused it to become AP-2 dependent. These results indicate that all three proteins have AP-dependent sorting signals, which may help to explain the relative abundance of AP complexes in CCVs. However, the relatively low abundance of cargo proteins in CCV preparations suggests either that some of the APs may be empty or that the preparations may be dominated by empty coats.
AP-1和AP-2复合物是网格蛋白包被囊泡（CCV）中最丰富的衔接蛋白，但在没有任何可检测到的AP-1或AP-2的情况下，网格蛋白介导的运输仍可发生。为了确定衔接蛋白的丰度是否反映货物的丰度，我们使用凝集素下拉法来鉴定来自人胎盘和大鼠肝脏的CCV中的主要膜糖蛋白。两种制剂都含有三种突出的高分子量蛋白：不依赖阳离子的甘露糖6-磷酸受体（CIMPR）、羧肽酶D（CPD）和低密度脂蛋白受体相关蛋白1（LRP1）。为了研究这些蛋白是如何被分选的，我们构建了CD8嵌合体并将其稳定转染到HeLa细胞中。CD8-CIMPR主要定位于早期/管状内体，CD8-CPD定位于反式高尔基体网络，CD8-LRP1定位于晚期/多囊泡内体。当通过siRNA耗尽网格蛋白时，所有三种构建体都重新分布到质膜。CD8-CIMPR也受到AP-2耗尽的强烈影响。CD8-CPD受到AP-2耗尽的中度影响，但同时耗尽AP-1和AP-2时受到强烈影响。CD8-LRP1仅受到AP-2耗尽的轻微影响；然而，突变LRP1尾部的NPXY基序会使其变得依赖AP-2。这些结果表明，所有三种蛋白都具有依赖AP的分选信号，这可能有助于解释CCV中AP复合物的相对丰度。然而，CCV制剂中货物蛋白的相对低丰度表明，要么一些AP可能是空的，要么制剂可能以空衣壳为主。
