Biology subjects

Spiess, M.

Publications and source records attributed to Spiess, M..

6 recordsLinked to original sources

bioRxiv2022

A common mechanism of Sec61 translocon inhibition by small molecules

The Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum (ER) membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural and synthetic small molecules specifically inhibit the Sec61 channel, generating cellular effects that are potentially useful for therapeutic purposes, but their inhibitory mechanisms remain unclear. Here we present near-atomic-resolution structures of the human Sec61 channel inhibited by a comprehensive panel of structurally distinct small molecules-- cotransin, decatransin, apratoxin F, ipomoeassin F, mycolactone, cyclotriazadisulfonamide (CADA) and eeyarestatin I (ESI). Remarkably, all inhibitors bind to a common lipid-exposed pocket formed by the partially open lateral gate and plug domain of the channel. Mutations conferring resistance to the inhibitors are clustered at this binding pocket. The structures indicate that Sec61 inhibitors stabilize the plug domain of Sec61 in a closed state, thereby preventing the protein-translocation pore from opening. Our study reveals molecular interactions between Sec61 and its inhibitors in atomic detail and offers the structural framework for further pharmacological studies and drug design.

biochemistry↗

bioRxiv2022

The small GTPase Arf1 regulates ATP synthesis and mitochondria homeostasis by modulating fatty acid metabolism

Lipid mobilization through fatty acid {beta}-oxidation is a central process essential for energy production during nutrient shortage. In yeast, this catabolic process starts in the peroxisome from where {beta}-oxidation products enter mitochondria and fuel the TCA cycle. Little is known about the physical and metabolic cooperation between these organelles. We found that expression of fatty acid transporters and of the rate-limiting enzyme involved in {beta}-oxidation are decreased in cells expressing a hyperactive mutant of the small GTPase Arf1, leading to an accumulation of fatty acids in lipid droplets. As a consequence, mitochondria became fragmented and ATP synthesis decreased. Genetic and pharmacological depletion of fatty acids phenocopied the arf1 mutant mitochondrial phenotype. Although {beta}-oxidation occurs mainly in mitochondria in mammals, Arf1s role in fatty acid metabolism is conserved. Together, our results indicate that Arf1 integrates metabolism into energy production by regulating fatty acid storage and utilization, and presumably organelle contact-sites.

cell biology↗

bioRxiv2021

Small disulfide loops in peptide hormones mediate self-aggregation and secretory granule sorting

Unlike constitutively secreted proteins, peptide hormones are stored in densely packed secretory granules, before regulated release upon stimulation. Secretory granules are formed at the trans-Golgi network (TGN) by self-aggregation of prohormones as functional amyloids. The nonapeptide hormone vasopressin, which forms a small disulfide loop, was shown to be responsible for granule formation of its precursor in the TGN as well as for toxic fibrillar aggregation of unfolded mutants in the endoplasmic reticulum (ER). Several other hormone precursors also contain similar small disulfide loops suggesting their function as a general device to mediate aggregation for granule biogenesis. To test this hypothesis, we studied the capacity of small disulfide loops of different hormone precursors to mediate aggregation in the ER and the TGN. They indeed induced ER aggregation although to different extents in Neuro-2a and COS-1 cells. Fused to a constitutively secreted reporter protein, they also promoted sorting into secretory granules, enhanced stimulated secretion, and increased Lubrol insolubility in AtT20 cells. These results support the hypothesis that small disulfide loops act as novel signals for secretory granule biogenesis and sorting by self-aggregation.

cell biology↗

bioRxiv2021

Shared and specific functions of Arfs 1-5 at the Golgi revealed by systematic knockouts

The ADP-ribosylation factors (Arfs) are small GTPases regulating membrane traffic in the secretory pathway. They are closely related and appear to have overlapping functions, regulators, and effectors. The functional specificity of individual Arfs and the extent of redundancy in vivo are still largely unknown. We addressed these questions by CRISPR/Cas9-mediated genomic deletion of the human class I (Arfs 1 and 3) and class II (Arfs 4 and 5) Arfs, either individually or in combination. Cells lacking individual Arfs or certain combinations were viable with only a slight growth defect when lacking Arf1 or Arf4. However, Arf1 and 4, and Arf4 and 5 could not be deleted simultaneously. Hence, class I Arfs are not essential and Arf4 alone was found to be sufficient for cell viability. Remarkably, two single knockouts produced specific and distinct phenotypes. Upon deletion of Arf1, the Golgi complex was enlarged and recruitment of vesicle coats decreased, confirming a major role of Arf1 in coat formation at the Golgi. Cell lines deleted for Arf4 exhibited secretion of ER resident proteins, indicating a specific defect in coatomer-dependent ER protein retrieval by the KDEL receptors. The knockout cell lines will be a useful tool to study other Arf-dependent processes.

cell biology↗

bioRxiv2020

Rabaptin5 targets autophagy to damaged early endosomes and Salmonella containing vacuoles by interaction with FIP200 and ATG16L1

Selective autophagy of damaged organelles is an important process for cellular homeostasis. The mechanisms how autophagy selects specific targets is often poorly understood. Rabaptin5 was previously known as a major regulator of early endosome identity and maturation. Here we identified two novel Rabaptin5 interactors: FIP200, a subunit of the ULK1 autophagy initiator complex, and ATG16L1, a central component of the E3-like enzyme in LC3 lipidation. Indeed, autophagy of early endosomes damaged by chloroquine or monensin treatment was found to require Rabaptin5 and particularly a specific short sequence motif binding to the WD domain of ATG16L1. Rabaptin5 and this interaction with ATG16L1 is further required for much of autophagic elimination of Salmonella enterica in phagosomes with early endosomal characteristics early after infection. Our results demonstrate a novel function of Rabaptin5 in quality control of early endosomes in the selective recruitment of autophagy to damaged early endosomes and phagosomes.

cell biology↗

bioRxiv2019

Retrograde transport of mannose-6-phosphate receptor depends on several sorting machineries as analyzed by sulfatable nanobodies

Retrograde protein transport from the cell surface and endosomes to the trans-Golgi network (TGN) is essential for membrane homeostasis in general and for the recycling of mannose-6-phosphate receptors (MPRs) for sorting of lysosomal hydrolases in particular. Several different sorting machineries have been implicated in retrieval from early or late endosomes to the TGN, mostly for the cation-independent MPR (CIMPR), mainly by analysis of steady-state localization and by interaction studies. We employed a nanobody-based sulfation tool to more directly determine transport kinetics from the plasma membrane to the TGN - the site of sulfation - for the cation-dependent MPR (CDMPR) with and without silencing of candidate machinery proteins. The clathrin adaptor AP-1 that operates bidirectionally at the TGN-to-endosome interface, which had been shown to cause reduced sulfation when rapidly depleted, produced hypersulfation of nanobodies internalized by CDMPR upon long-term silencing, reflecting accumulation in the TGN. In contrast, knockdown of retromer (Vps26), epsinR, or Rab9 reduced CDMPR arrival to the TGN. No effect was observed upon silencing of TIP47. Most surprisingly, depletion of the GGA (Golgi-localized, {gamma}-adaptin ear-containing, Arf-binding) proteins inhibited retrograde transport rather than TGN exit. This study illustrates the usefulness of derivatized, sulfation-competent nanobodies to analyze retrograde protein transport to identify the contributions of different machineries.

cell biology↗