Role of sedlin, a TRAPP complex subunit, in membrane trafficking and in the pathogenesis of Spondyleopyphiseal Dysplasia Tarda

Genetic defects occurring in the sedlin gene, a conserved component of TRAPP complex, cause Spondyloepiphyseal Dysplasia Tarda (SEDT), a rare progressive condition characterised by impaired chondrogenesis resulting in short stature, flattening of the vertebrae, and premature osteoarthritis. The role of sedlin in the pathogenesis of SEDT disease so far is still unknown. Prompted by the consideration that sedlin is ubiquitously expressed but that sedlin mutations cause cartilaginous-restricted dysfunctions, I hypothesized that sedlin might exert a role in membrane trafficking generally but in particular in the transport of chondrocyte- specific cargoes, such as type II procollagen (PCII). This hypothesis was reinforced by the fact that mutations in PCII give rise to autosomal dominant forms of spondyloepiphiseal dysplasia. I tested this hypothesis by analyzing the involvement of sedlin in the transport of different classes of secretory cargoes and found that sedlin is selectively required for PCII to exit the ER, while it is not essential for ER exit of small soluble and membrane-associated cargoes. I have also identified the molecular mechanism underlying this role of sedlin in its ability to bind the GTPase Sarl and to control the membrane-cytosol cycle of Sarl itself and of the COPIl coat complex at the level of the ER exit sites. Sedlin depletion and/or mutation in SEDT patients slows down the Sar1 cycle and prolongs the membrane association of Sar1-GTP at the ER exit sites, thus inducing constriction and premature fission of nascent carriers which fail to incorporate the large PC protofibrils but are still competent for smaller secretory cargoes. All together these findings provide new insights not only into understanding the role of sedlin but also shed new light on the molecular mechanisms underlying the onset of the SEDT disease

A novel class of small repetitive DNA sequences in<i>Enterococcus faecalis</i>

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The activity of Sac1 across ER-TGN contact sites requires the four-phosphate-adaptor-protein-1

Phosphatidylinositol-4-phosphate (PI4P), a phosphoinositide with key roles in the Golgi complex, is made by Golgi-associated phosphatidylinositol-4 kinases and consumed by the 4-phosphatase Sac1 that, instead, is an ER membrane protein. Here, we show that the contact sites between the ER and the TGN (ERTGoCS) provide a spatial setting suitable for Sac1 to dephosphorylate PI4P at the TGN. The ERTGoCS, though necessary, are not sufficient for the phosphatase activity of Sac1 on TGN PI4P, since this needs the phosphatidyl-four-phosphate-adaptor-protein-1 (FAPP1). FAPP1 localizes at ERTGoCS, interacts with Sac1, and promotes its in-trans phosphatase activity in vitro. We envision that FAPP1, acting as a PI4P detector and adaptor, positions Sac1 close to TGN domains with elevated PI4P concentrations allowing PI4P consumption. Indeed, FAPP1 depletion induces an increase in TGN PI4P that leads to increased secretion of selected cargoes (e.g., ApoB100), indicating that FAPP1, by controlling PI4P levels, acts as a gatekeeper of Golgi exit.Peer reviewe

Molecular determinants of ER-Golgi contacts identified through a new FRET-FLIM system

ER-TGN contact sites (ERTGoCS) have been visualized by electron microscopy, but their location in the crowded perinuclear area has hampered their analysis via optical microscopy as well as their mechanistic study. To overcome these limits we developed a FRET-based approach and screened several candidates to search for molecular determinants of the ERTGoCS. These included the ER membrane proteins VAPA and VAPB and lipid transfer proteins possessing dual (ER and TGN) targeting motifs that have been hypothesized to contribute to the maintenance of ERTGoCS, such as the ceramide transfer protein CERT and several members of the oxysterol binding proteins. We found that VAP proteins, OSBP1, ORP9, and ORP10 are required, with OSBP1 playing a redundant role with ORP9, which does not involve its lipid transfer activity, and ORP10 being required due to its ability to transfer phosphatidylserine to the TGN. Our results indicate that both structural tethers and a proper lipid composition are needed for ERTGoCS integrity.Peer reviewe

ER exit sites take the strain

Cells are able to adapt their growth to external mechanical strain. A recent study by Phuyal et al (2022) has shown that these responses depend on the heterodimerization of two small GTPases

Exiting the ER: What we know and what we don't

The vast majority of proteins that are transported to different cellular compartments and secreted from the cell require coat protein complex II (COPII) for export from the endoplasmic reticulum (ER). Many of the molecular mechanisms underlying COPII assembly are understood in great detail, but it is becoming increasingly evident that this basic machinery is insufficient to account for diverse aspects of protein export from the ER that are observed in vivo. Here we review recent data that have furthered our mechanistic understanding of COPII assembly and, in particular, how genetic diseases associated with the early secretory pathway have added fundamental insights into the regulation of ER-derived carrier formation. We also highlight some unresolved issues that future work should address to better understand the physiology of COPII-mediated transport

TRAPPing Rab18 in lipid droplets

A number of membrane trafficking components are associated with lipid droplets (LDs) and/or are involved in their biogenesis. In this issue of The EMBO Journal, Li et al (2016) show that the mammalian TRAPPII (TRAnsport Protein Particle) complex acts as an LD-associated GEF for Rab18, thereby regulating LD homeostasis. © 2017 EMBO

ER-Golgi membrane contact sites

Membrane contact sites (MCSs) are sites where the membranes of two different organelles come into close apposition (10-30 nm). Different classes of proteins populate MCSs including factors that act as tethers between the two membranes, proteins that use the MCSs for their function (mainly lipid or ion exchange), and regulatory proteins and enzymes that can act in trans across the MCSs. The ER-Golgi MCSs were visualized by electron microscopists early in the sixties but have remained elusive for decades due to a lack of suitable methodological approaches. Here we report recent progress in the study of this class of MCSs that has led to the identification of their main morphological features and of some of their components and roles. Among these, lipid transfer proteins and lipid exchange have been the most studied and understood so far. However, many unknowns remain regarding their regulation and their role in controlling key TGN functions such as sorting and trafficking as well as their relevance in physiological and pathological conditions