Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, United States; Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA 20147, United States.
Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, United States.
Matrix Biol. 2020 Nov;93:79-94. doi: 10.1016/j.matbio.2020.06.002. Epub 2020 Jun 17.
Efficient quality control and export of procollagen from the cell is crucial for extracellular matrix homeostasis, yet it is still incompletely understood. One of the debated questions is the role of a collagen-specific ER chaperone HSP47 in these processes. Most ER chaperones preferentially bind to unfolded polypeptide chains, enabling selective export of natively folded proteins from the ER after chaperone release. In contrast, HSP47 preferentially binds to the natively folded procollagen and is believed to be released only in the ER-Golgi intermediate compartment (ERGIC) or cis-Golgi. HSP47 colocalization with procollagen in punctate structures observed by immunofluorescence imaging of fixed cells has thus been interpreted as evidence for HSP47 export from the ER together with procollagen in transport vesicles destined for ERGIC or Golgi. To understand the mechanism of this co-trafficking and its physiological significance, we imaged the dynamics of fluorescently tagged type I procollagen and HSP47 punctate structures in live MC3T3 murine osteoblasts with up to 120 nm spatial and 500 ms time resolution. Contrary to the prevailing model, we discovered that most bona fide carriers delivering procollagen from ER exit sites (ERESs) to Golgi contained no HSP47, unless the RDEL signal for ER retention in HSP47 was deleted or mutated. These transport intermediates exhibited characteristic rapid, directional motion along microtubules, while puncta with colocalized HSP47 and procollagen similar to the ones described before had only limited, stochastic motion. Live cell imaging and fluorescence recovery after photobleaching revealed that the latter puncta (including the ones induced by ARF1 inhibition) were dilated regions of ER lumen, ERESs, or autophagic structures surrounded by lysosomal membranes. Procollagen was colocalized with HSP47 and ERGIC53 at ERESs. It was colocalized with ERGIC53 but not HSP47 in Golgi-bound transport intermediates. Our results suggest that procollagen and HSP47 sorting occurs at ERES before procollagen is exported from the ER in Golgi-bound transport intermediates, providing new insights into mechanisms of procollagen trafficking.
胶原蛋白从细胞中的有效质量控制和输出对于细胞外基质的动态平衡至关重要，但人们对此仍不完全了解。其中一个有争议的问题是胶原蛋白特异性内质网伴侣 HSP47 在这些过程中的作用。大多数内质网伴侣优先结合未折叠的多肽链，从而能够在伴侣释放后选择性地将天然折叠的蛋白质从内质网中输出。相比之下，HSP47 优先结合天然折叠的前胶原蛋白，并且据信仅在内质网-高尔基体中间隔室（ERGIC）或顺式高尔基体中释放。免疫荧光成像固定细胞中观察到的前胶原蛋白与 HSP47 在点状结构中的共定位因此被解释为 HSP47 与前胶原蛋白一起从内质网中输出的证据，它们在运往 ERGIC 或高尔基体的运输小泡中。为了了解这种共运输的机制及其生理意义，我们使用高达 120nm 的空间分辨率和 500ms 的时间分辨率，对活的 MC3T3 鼠成骨细胞中荧光标记的 I 型前胶原蛋白和 HSP47 点状结构的动力学进行了成像。与流行的模型相反，我们发现，除非删除或突变 HSP47 中用于内质网保留的 RDEL 信号，否则大多数从内质网出口部位（ERES）向高尔基体运送前胶原蛋白的真正载体都不含 HSP47。这些运输中间体表现出沿着微管快速、定向运动的特征，而与之前描述的类似的具有共定位 HSP47 和前胶原蛋白的点状结构仅具有有限的、随机的运动。活细胞成像和光漂白后荧光恢复显示，后一种点状结构（包括由 ARF1 抑制诱导的点状结构）是内质网腔、ERES 或被溶酶体膜包围的自噬结构的扩张区域。前胶原蛋白与 HSP47 和 ERGIC53 在 ERES 处共定位。它与 ERGIC53 共定位，但不在高尔基体结合的运输中间体中与 HSP47 共定位。我们的结果表明，在前胶原蛋白在高尔基体结合的运输中间体中从内质网中输出之前，胶原蛋白和 HSP47 的分拣发生在 ERES 处，这为前胶原蛋白运输的机制提供了新的见解。
