The majority of all proteins expressed in the human osteosarcoma cell line U2OS were absolutely quantified by mass spectrometry. The quantified proteins span a concentration range of seven orders of magnitude up to 20 000 000 copies per cell.

SummaryNuclear pore complexes (NPCs) are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Elucidating their 110 MDa structure imposes a formidable challenge and requires in situ structural biology approaches. Fifteen out of about thirty nucleoporins (Nups) are structured and form the Y- and inner ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ∼60 nm in diameter 1. The scaffold is decorated with transport channel Nups that often contain phenylalanine (FG)-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y-complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here, we combined cryo electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modeling to generate the most comprehensive architectural model of the NPC to date. Our data suggest previously unknown protein interfaces across Y-complexes and to inner ring complex members. We demonstrate that the higher eukaryotic transport channel Nup358 (RanBP2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport channel Nups. We conclude that, similarly to coated vesicles, multiple copies of the same structural building block - although compositionally identical - engage in different local sets of interactions and conformations.

The nuclear pore complex (NPC) is a fundamental component of all eukaryotic cells that facilitates nucleocytoplasmic exchange of macromolecules. It is assembled from multiple copies of about 30 nucleoporins. Due to its size and complex composition, determining the structure of the NPC is an enormous challenge, and the overall architecture of the NPC scaffold remains elusive. In this study, we have used an integrated approach based on electron tomography, single-particle electron microscopy, and crosslinking mass spectrometry to determine the structure of a major scaffold motif of the human NPC, the Nup107 subcomplex, in both isolation and integrated into the NPC. We show that 32 copies of the Nup107 subcomplex assemble into two reticulated rings, one each at the cytoplasmic and nuclear face of the NPC. This arrangement may explain how changes of the diameter are realized that would accommodate transport of huge cargoes.

The stoichiometry of the human nuclear pore complex is revealed by targeted mass spectrometry and super-resolution microscopy. The analysis reveals that the composition of the nuclear pore and other nuclear protein complexes is remodeled as a function of the cell type.

The lysosome degrades and recycles macromolecules, signals to the master growth regulator mTORC1 [mechanistic target of rapamycin (mTOR) complex 1], and is associated with human disease. We performed quantitative proteomic analyses of rapidly isolated lysosomes and found that nutrient levels and mTOR dynamically modulate the lysosomal proteome. Upon mTORC1 inhibition, NUFIP1 (nuclear fragile X mental retardation-interacting protein 1) redistributes from the nucleus to autophagosomes and lysosomes. Upon these conditions, NUFIP1 interacts with ribosomes and delivers them to autophagosomes by directly binding to microtubule-associated proteins 1A/1B light chain 3B (LC3B). The starvation-induced degradation of ribosomes via autophagy (ribophagy) depends on the capacity of NUFIP1 to bind LC3B and promotes cell survival. We propose that NUFIP1 is a receptor for the selective autophagy of ribosomes.

A large body of evidence supports the involvement of heparan sulfate (HS) proteoglycans in physiological processes such as development and diseases including cancer and neurodegenerative disorders. The role of HS emerges from its ability to interact and regulate the activity of a vast number of extracellular proteins including growth factors and extracellular matrix components. A global view on how protein-HS interactions influence the extracellular proteome and, consequently, cell function is currently lacking. Here, we systematically investigate the functional and structural properties that characterize HS-interacting proteins and the network they form. We collected 435 human proteins interacting with HS or the structurally related heparin by integrating literature-derived and affinity proteomics data. We used this data set to identify the topological features that distinguish the heparin/HS-interacting network from the rest of the extracellular proteome and to analyze the enrichment of gene ontology terms, pathways, and domain families in heparin/HS-binding proteins. Our analysis revealed that heparin/ HS-binding proteins form a highly interconnected network, which is functionally linked to physiological and pathological processes that are characteristic of higher organisms. Therefore, we then investigated the existence of a correlation between the expansion of domain families characteristic of the heparin/HS interactome and the increase in biological complexity in the metazoan lineage. A strong positive correlation between the expansion of the heparin/HS interactome and biosynthetic machinery and organism complexity emerged. The evolutionary role of HS was reinforced by the presence of a rudimentary HS biosynthetic machinery in a unicellular organism at the root of the metazoan lineage.

SummaryAging is associated with the decline of protein, cell, and organ function. Here, we use an integrated approach to characterize gene expression, bulk translation, and cell biology in the brains and livers of young and old rats. We identify 468 differences in protein abundance between young and old animals. The majority are a consequence of altered translation output, that is, the combined effect of changes in transcript abundance and translation efficiency. In addition, we identify 130 proteins whose overall abundance remains unchanged but whose sub-cellular localization, phosphorylation state, or splice-form varies. While some protein-level differences appear to be a generic property of the rats’ chronological age, the majority are specific to one organ. These may be a consequence of the organ’s physiology or the chronological age of the cells within the tissue. Taken together, our study provides an initial view of the proteome at the molecular, sub-cellular, and organ level in young and old rats.

The cell-extracellular matrix interface is a crowded space whose structure is dependent on macromolecular assemblies that are dynamic in time, molecular composition and location. Signals travel from one cell to another (or to the same cell) by the regulated assembly/disassembly of molecular complexes. These signals can evoke relatively simple biological responses such cell proliferation and migration, but once integrated, they guide cell fate in complex biological phenomena such as embryonic development and organism homeostasis. Heparan sulfate proteoglycans are ubiquitous components of this space and important actors of these processes in all tissue-organized life forms. A key feature of heparan sulfate is its size, 40 nm to 160 nm, which enables it to integrate self-assembling macromolecular structures over substantial length scales. What is the structure of heparan sulfate? Why do we think heparan sulfate is so important? How do we try to explain its activity? What do we know about its interactions? These questions together with a final look to the future are the "menu" of this review.