Human immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrated that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, cone-shaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle.

Quantitative mass spectrometry has established proteome-wide regulation of protein abundance and post-translational modifications in various biological processes. Here, we used quantitative mass spectrometry to systematically analyze the thermal stability and solubility of proteins on a proteome-wide scale during the eukaryotic cell cycle. We demonstrate pervasive variation of these biophysical parameters with most changes occurring in mitosis and G1. Various cellular pathways and components vary in thermal stability, such as cell-cycle factors, polymerases, and chromatin remodelers. We demonstrate that protein thermal stability serves as a proxy for enzyme activity, DNA binding, and complex formation in situ. Strikingly, a large cohort of intrinsically disordered and mitotically phosphorylated proteins is stabilized and solubilized in mitosis, suggesting a fundamental remodeling of the biophysical environment of the mitotic cell. Our data represent a rich resource for cell, structural, and systems biologists interested in proteome regulation during biological transitions.

Eukaryotic 60S ribosomal subunits are comprised of three rRNAs and ∼50 ribosomal proteins. The initial steps of their formation take place in the nucleolus, but, owing to a lack of structural information, this process is poorly understood. Using cryo-EM, we solved structures of early 60S biogenesis intermediates at 3.3 Å to 4.5 Å resolution, thereby providing insights into their sequential folding and assembly pathway. Besides revealing distinct immature rRNA conformations, we map 25 assembly factors in six different assembly states. Notably, the Nsa1-Rrp1-Rpf1-Mak16 module stabilizes the solvent side of the 60S subunit, and the Erb1-Ytm1-Nop7 complex organizes and connects through Erb1's meandering N-terminal extension, eight assembly factors, three ribosomal proteins, and three 25S rRNA domains. Our structural snapshots reveal the order of integration and compaction of the six major 60S domains within early nucleolar 60S particles developing stepwise from the solvent side around the exit tunnel to the central protuberance.

The molecular events that direct nuclear pore complex (NPC) assembly toward nuclear envelopes have been conceptualized in two pathways that occur during mitosis or interphase, respectively. In gametes and embryonic cells, NPCs also occur within stacked cytoplasmic membrane sheets, termed annulate lamellae (AL), which serve as NPC storage for early development. The mechanism of NPC biogenesis at cytoplasmic membranes remains unknown. Here, we show that during Drosophila oogenesis, Nucleoporins condense into different precursor granules that interact and progress into NPCs. Nup358 is a key player that condenses into NPC assembly platforms while its mRNA localizes to their surface in a translation-dependent manner. In concert, Microtubule-dependent transport, the small GTPase Ran and nuclear transport receptors regulate NPC biogenesis in oocytes. We delineate a non-canonical NPC assembly mechanism that relies on Nucleoporin condensates and occurs away from the nucleus under conditions of cell cycle arrest.

Nuclear pore complexes (NPCs) span the nuclear envelope (NE) and mediate nucleocytoplasmic transport. In metazoan oocytes and early embryos, NPCs reside not only within the NE, but also at some endoplasmic reticulum (ER) membrane sheets, termed annulate lamellae (AL). Although a role for AL as NPC storage pools has been discussed, it remains controversial whether and how they contribute to the NPC density at the NE. Here, we show that AL insert into the NE as the ER feeds rapid nuclear expansion in Drosophila blastoderm embryos. We demonstrate that NPCs within AL resemble pore scaffolds that mature only upon insertion into the NE. We delineate a topological model in which NE openings are critical for AL uptake that nevertheless occurs without compromising the permeability barrier of the NE. We finally show that this unanticipated mode of pore insertion is developmentally regulated and operates prior to gastrulation.

Gene expression is a coordinated multistep process that begins with transcription and RNA processing in the nucleus followed by mRNA export to the cytoplasm for translation. Here we report the identification of a protein, Sus1, which functions in both transcription and mRNA export. Sus1 is a nuclear protein with a concentration at the nuclear pores. Biochemical analyses show that Sus1 interacts with SAGA, a large intranuclear histone acetylase complex involved in transcription initiation, and with the Sac3-Thp1 complex, which functions in mRNA export with specific nuclear pore proteins at the nuclear basket. DNA macroarray analysis revealed that Sus1 is required for transcription regulation. Moreover, chromatin immunoprecipitation showed that Sus1 is associated with the promoter of a SAGA-dependent gene during transcription activation. Finally, mRNA export is impaired in sus1 mutants. These data provide an unexpected connection between the SAGA histone acetylase complex and the mRNA export machinery.

Proteotypes, like genotypes, have been found to vary between individuals in several studies, but consistent molecular functional traits across studies remain to be quantified. In a meta-analysis of 11 proteomics datasets from humans and mice, we use co-variation of proteins in known functional modules across datasets and individuals to obtain a consensus landscape of proteotype variation. We find that individuals differ considerably in both protein complex abundances and stoichiometry. We disentangle genetic and environmental factors impacting these metrics, with genetic sex and specific diets together explaining 13.5% and 11.6% of the observed variation of complex abundance and stoichiometry, respectively. Sex-specific differences, for example, include various proteins and complexes, where the respective genes are not located on sex-specific chromosomes. Diet-specific differences, added to the individual genetic backgrounds, might become a starting point for personalized proteotype modulation toward desired features.