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.

有效的质量控制和细胞前胶原的输出对于细胞外基质稳态至关重要，但仍不完全清楚。有争议的问题之一是胶原蛋白特异性 ER 伴侣 HSP47 在这些过程中的作用。大多数 ER 伴侣优先结合未折叠的多肽链，从而在伴侣释放后选择性地从 ER 中输出天然折叠的蛋白质。相比之下，HSP47 优先与天然折叠的前胶原结合，并且被认为仅在 ER-高尔基中间室 (ERGIC) 或顺式-高尔基中释放。因此，通过固定细胞的免疫荧光成像观察到的点状结构中的 HSP47 与前胶原共定位已被解释为 HSP47 从 ER 输出的证据，以及运往 ERGIC 或高尔基体的运输囊泡中的前胶原。为了了解这种共同贩运的机制及其生理意义，我们以高达 120 nm 的空间分辨率和 500 ms 的时间分辨率对活 MC3T3 小鼠成骨细胞中荧光标记的 I 型前胶原和 HSP47 点状结构的动力学进行了成像。与流行模型相反，我们发现大多数真正的载体将前胶原从 ER 出口部位 (ERES) 运送到高尔基体不含 HSP47，除非 HSP47 中 ER 保留的 RDEL 信号被删除或突变。这些转运中间体表现出特征性的沿微管的快速定向运动，而与之前描述的相似的共定位 HSP47 和前胶原的斑点仅具有有限的随机运动。光漂白后的活细胞成像和荧光恢复表明，后者的斑点（包括由 ARF1 抑制诱导的斑点）是 ER 腔、ERES 或被溶酶体膜包围的自噬结构的扩张区域。前胶原在 ERES 与 HSP47 和 ERGIC53 共定位。它在高尔基结合的运输中间体中与 ERGIC53 而不是 HSP47 共定位。我们的研究结果表明，前胶原和 HSP47 分选发生在 ERES，然后前胶原从高尔基体结合的运输中间体中的 ER 输出，为前胶原运输机制提供了新的见解。它在高尔基结合的运输中间体中与 ERGIC53 而不是 HSP47 共定位。我们的研究结果表明，前胶原和 HSP47 分选发生在 ERES，然后前胶原从高尔基体结合的运输中间体中的 ER 输出，为前胶原运输机制提供了新的见解。它在高尔基结合的运输中间体中与 ERGIC53 而不是 HSP47 共定位。我们的研究结果表明，前胶原和 HSP47 分选发生在 ERES，然后前胶原从高尔基体结合的运输中间体中的 ER 输出，为前胶原运输机制提供了新的见解。