TFG Promotes Organization of Transitional ER and Efficient Collagen Secretion

Collagen is the most abundant protein in the animal kingdom. It is of fundamental importance during development for cell differentiation and tissue morphogenesis as well as in pathological processes such as fibrosis and cancer cell migration. However, our understanding of the mechanisms of procollagen secretion remains limited. Here, we show that TFG organizes transitional ER (tER) and ER exit sites (ERESs) into larger structures. Depletion of TFG results in dispersion of tER elements that remain associated with individual ER-Golgi intermediate compartments (ERGICs) as largely functional ERESs. We show that TFG is not required for the transport and packaging of small soluble cargoes but is necessary for the export of procollagen from the ER. Our work therefore suggests a key relationship between the structure and function of ERESs and a central role for TFG in optimizing COPII assembly for procollagen export.Medical Research Council UK/MR/J000604/1Medical Research Council UK/MR/K018019/1Medical Research Council UK/MR/G080184

A general role for TANGO1, encoded by MIA3, in secretory pathway organization and function

Complex machinery is required to drive secretory cargo export from the endoplasmic reticulum (ER), which is an essential process in eukaryotic cells. In vertebrates, the MIA3 gene encodes two major forms of transport and Golgi organization protein 1 (TANGO1S and TANGO1L), which have previously been implicated in selective trafficking of procollagen. Using genome engineering of human cells, light microscopy, secretion assays, genomics and proteomics, we show that disruption of the longer form, TANGO1L, results in relatively minor defects in secretory pathway organization and function, including having limited impacts on procollagen secretion. In contrast, loss of both long and short forms results in major defects in cell organization and secretion. These include a failure to maintain the localization of ERGIC53 (also known as LMAN1) and SURF4 to the ER–Golgi intermediate compartment and dramatic changes to the ultrastructure of the ER–Golgi interface. Disruption of TANGO1 causes significant changes in early secretory pathway gene and protein expression, and impairs secretion not only of large proteins, but of all types of secretory cargo, including small soluble proteins. Our data support a general role for MIA3/TANGO1 in maintaining secretory pathway structure and function in vertebrate cells

Regulator of calcineurin-2 is a centriolar protein with a role in cilia length control

Almost every cell in the human body extends a primary cilium. Defective cilia function leads to a set of disorders known as ciliopathies characterised by debilitating developmental defects affecting many tissues. Here we report a new role for regulator of calcineurin 2, RCAN2, in primary cilia function. It localises to centrioles and the basal body and is required to maintain normal cilia length. RCAN2 was identified as the most strongly upregulated gene from a comparative RNAseq analysis of cells in which expression of the Golgi matrix protein giantin had been abolished by gene editing. In contrast to previous work where we showed that depletion of giantin by RNAi results in defects in ciliogenesis and in cilia length control, giantin knockout cells generate normal cilia on serum withdrawal. Furthermore, giantin knockout zebrafish show increased expression of RCAN2. Importantly, suppression of RCAN2 expression in giantin knockout cells results in the same defects in cilia length control seen on RNAi of giantin itself. Together these data define RCAN2 as a regulator of cilia function that can compensate for loss of giantin function.</jats:p

P4HB recurrent missense mutation causing Cole-Carpenter syndrome

BACKGROUND: Cole-Carpenter syndrome (CCS) is commonly classified as a rare Osteogenesis Imperfecta (OI) disorder. This was following the description of two unrelated patients with very similar phenotypes who were subsequently shown to have a heterozygous missense mutation in P4HB. OBJECTIVES: Here, we report a 3-year old female patient with severe OI who on exome sequencing was found to carry the same missense mutation in P4HB as reported in the original cohort. We discuss the genetic heterogeneity of CCS and underlying mechanism of P4HB in collagen production. METHODS: We undertook detailed clinical, radiological and molecular phenotyping in addition, to analysis of collagen in cultured fibroblasts and electron microscopic examination in the patient reported here. RESULTS: The clinical phenotype appears consistent in patients reported so far but interestingly, there also appears to be a definitive phenotypic clue (crumpling metadiaphyseal fractures of the long tubular bones with metaphyseal sclerosis which are findings that are uncommon in OI) to the underlying genotype (P4HB variant). DISCUSSION: P4HB (Prolyl 4-hydroxylase, betasubunit) encodes for PDI (Protein Disulfide isomerase) and in cells, in its tetrameric form, catalyses formation of 4-hydroxyproline in collagen. The recurrent variant in P4HB, c.1178A>G, p.Tyr393Cys, sits in the C-terminal reactive centre and is said to interfere with disulphide isomerase function of the C-terminal reactive centre. P4HB catalyses the hydroxylation of proline residues within the X-Pro-Gly repeats in the procollagen helical domain. Given the inter-dependence of extracellular matrix (ECM) components in assembly of a functional matrix, our data suggest that it is the organisation and assembly of the functional ECM that is perturbed rather than the secretion of collagen type I per se. CONCLUSIONS: We provide additional evidence of P4HB as a cause of a specific form of OI-CCS and expand on response to treatment with bisphosphonates in this rare disorder

ER-to-Golgi trafficking of procollagen in the absence of large carriers

AbstractSecretion and assembly of collagen is fundamental to the function of the extracellular matrix. Defects in the assembly of a collagen matrix lead to pathologies including fibrosis and osteogenesis imperfecta. Owing to the size of fibril-forming procollagen molecules it is assumed that they are transported from the endoplasmic reticulum to the Golgi in specialised large COPII-dependent carriers. Here, analysing endogenous procollagen and a new engineered GFP-tagged form, we show that transport to the Golgi occurs in the absence of large carriers. Large GFP-positive structures are observed occasionally but these are non-dynamic, are not COPII-positive, and label with markers of the ER. We propose a “short-loop” model of COPII-dependent ER-to-Golgi traffic that, while consistent with models of ERGIC-dependent expansion of COPII carriers, does not invoke long-range trafficking of large vesicular structures. Our findings provide an important insight into the process of procollagen trafficking and reveal a short-loop pathway from the ER to the Golgi, without the use of large carriers.SummaryTrafficking of procollagen is essential for normal cell function. Here, imaging of GFP-tagged type I procollagen reveals that it is transported from the endoplasmic reticulum to the Golgi, without the use of large carriers.</jats:sec

A general role for MIA3/TANGO1 in secretory pathway organization and function

Raw data associated with a manuscript describing engineered knockout of Mia3/TANGO1 in human cells