Elsevier

Matrix Biology

Volume 93, November 2020, Pages 79-94
Matrix Biology

Mechanisms of procollagen and HSP47 sorting during ER-to-Golgi trafficking

https://doi.org/10.1016/j.matbio.2020.06.002Get rights and content
Under a Creative Commons license
open access

Highlights

  • 120 nm, 500 ms/frame imaging of live osteoblasts identifies ER-Golgi procollagen carriers.

  • ER-Golgi procollagen carriers contain ERGIC protein ERGIC53 but not HSP47.

  • HSP47 is released from procollagen at ER exit sites rather than ERGIC or cis-Golgi.

  • Procollagen carrier formation requires GDP to GTP exchange at ARF1 GTPase.

Abstract

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.

Keywords

Collagen
HSP47
trafficking
ER exit sites
live cell imaging

Abbreviations

ER
Endoplasmic Reticulum
ERES
ER exit site
ERGIC
ER Golgi Intermediate Compartment
BFA
Brefeldin A
FP
fluorescent protein
FRAP
Fluorescence Recovery After Photobleaching

Cited by (0)