Ilana Berlin

In this study, we sought to comprehensively map ubiquitin signaling networks at the crossroads of cellular proliferation and proteostasis, both crucial in cancer. Genome-wide CRISPR knockout screening in the presence of the ubiquitin E1 inhibitor TAK-243 identified 239 genes whose ablation sensitizes cells to ubiquitination blockade. Follow-up studies reveal a role for the nuclear protein, C16orf72/HECT, UBA, and WWE domain containing E3 ubiquitin protein ligase 1 (HUWE1)–Associated Protein modifying Stress Responses (HAPSTR1), in chromatin proteostasis and DNA replication. We show that HAPSTR1 recruits the E3 ligase HUWE1 to the chromatin, keeping the chromatin-associated proteome in check, and is itself turned over by HUWE1 during cell cycle progression. Structural modeling of the HAPSTR1/HUWE1 complex, supported by mutational analysis, demonstrates that loss of HUWE1 binding renders HAPSTR1 unable to support cellular proliferation when ubiquitin signaling is suppressed. Consequently, HAPSTR1 ablation impairs clearance of chromatin-associated proteins, triggering global transcriptional and proteostatic adaptations. Under diminished ubiquitin signaling, these adaptive mechanisms give way to replication stress and premature S phase entry, culminating in cell cycle collapse and DNA damage. Our findings uncover protein networks exhibiting synthetic lethality with ubiquitin signaling blockade and highlight vulnerabilities of actively proliferating cells to proteostatic imbalance at the chromatin.

To integrate diverse cellular function and coordinate homeostasis, the endoplasmic reticulum (ER) communicates with all other intracellular compartments through physical interfaces termed membrane contact sites (MCSs). Given the sheer diversity of possible MCS pairings, how the ER discriminates between simultaneously available binding partners remains unclear. To explore this, we performed comparative endogenous contact site mapping for three closely related VAP family tethers and uncovered a unique set of FFAT motif selectivity and MCS footprints for each: VAPA at mitochondria and TGN, VAPB at mitochondria and peroxisomes, and MOSPD2 at mature endosomes, lysosomes and lipid droplets. In line with their MCS selectivity profiles, co-depletion of VAPA and VAPB, but not MOSPD2, caused systemic breakdown in mitochondrial integrity, while silencing of MOSPD2 alone was sufficient to disrupt the organization and transport of (endo)lysosomes. Unexpectedly, coincident loss of VAPA and VAPB instigated ER contact site rewiring by redirecting endogenous MOSPD2 to the collapsing mitochondrial network. Our findings define hierarchies of ER contact site formation and reveal compensation mechanisms exploited by cells to safeguard organelle homeostasis and crosstalk.

Many intracellular bacterial pathogens establish membrane-bound niches derived from host material to survive and replicate within host cells. These compartments are carved through subversion of host intracellular trafficking pathways to modulate the composition of the vacuolar membrane. Although recycling-associated small Rab GTPases are frequently observed at infection sites, their role in membrane remodeling and cargo sorting has remained poorly understood. Here, we leverage the extensive early membrane remodeling occurring during Salmonella infection to dissect this process. Using endogenously tagged cell lines, we characterize the dynamics of the recycling small GTPases Rab14 and Rab11 at Salmonella intracellular infection sites. We report that Rab14 recruits its effector Rufy1, which mediates sorting of CI-M6PR through recycling tubules. Subsequently, Rab11 disperses the newly formed recycling endosomes by interacting with its effector Fip3 for retrograde transport. This pathway is controlled by the deubiquitinating enzyme USP32, which targets both Rab14 and Rab11, enhancing their interactions with Rufy1 and Fip3, respectively. We further show that USP32 is recruited to the infection site via local enrichment of phosphatidylserine-positive membranes, a process triggered by the Salmonella effector proteins SopE/E2. Importantly, silencing USP32 significantly impairs intracellular bacterial survival. Our findings reveal a remarkable exploitation of host signaling and trafficking pathways by Salmonella to construct a pro-replicative niche. Targeting these pathways may offer new strategies for therapeutic intervention against intracellular bacterial infections.

Execution of all cellular functions depends on a healthy proteome, whose maintenance requires multimodal oversight. Roughly a third of human proteins reside in membranes and thus present unique topological challenges with respect to biogenesis and degradation. To meet these challenges, eukaryotes have evolved organellar pathways of protein folding and quality control. Most transmembrane proteins originate in the endoplasmic reticulum (ER), where they are subject to surveillance and, if necessary, removal through either ER-associated proteasomal degradation (cytosolic pathway) or selective autophagy (ER-phagy; organellar pathway). In the latter case, ER cargoes are shuttled to (endo)lysosomes – the same organelles that degrade cell surface molecules via endocytosis. Here, we provide an overview of dynamic coordination between the ER and endolysosomes, with a focus on their engagement in specialized physical interfaces termed membrane contact sites (MCSs). We cover how cross-compartmental integration through MCSs allows biosynthetic and proteolytic organelles to fine-tune each other's membrane composition, organization, and dynamics and facilitates recovery from proteotoxic stress. Along the way, we highlight recent developments and open questions at the crossroads between organelle biology and protein quality control and cast them against the backdrop of factor-specific diseases associated with perturbed membrane homeostasis.

Extracellular vesicles (EVs) are crucial mediators of intercellular communication that originate through one of two pathways: outward budding of the plasma membrane (PM) or fusion of mature (late) endosomes with the PM. How cells balance these EV biogenesis routes remains unclear. To address this, we performed a genome-wide CRISPR activation screen for genes that increase cell surface levels of the tetraspanin CD63—an EV marker that shuttles between late endosomes and the PM. This unbiased approach identified a membrane adaptor protein, MARCKSL1, commonly upregulated in diverse tumor types. Follow-up studies, integrating genomic activation/ablation with microscopic and proteomic approaches, revealed that MARCKSL1 potentiates EV secretion from the PM, (in part) at the expense of late endosome—PM fusion. Probing the molecular context of MARCKSL1 function, we implicate PM-bridging cytoskeletal components (e.g., Radixin) and SNARE-associated proteins (e.g., STXBP3) as collaborators of MARCKSL1. Collectively, our findings reveal new mechanistic underpinnings of PM remodeling and position MARCKSL1 as a gauge between different platforms of EV biogenesis.

Proteotoxic stress causes profound endoplasmic reticulum (ER) membrane remodeling into a perinuclear quality control compartment (ERQC) for the degradation of misfolded proteins. Subsequent return to homeostasis involves clearance of the ERQC by endolysosomes. However, the factors that control perinuclear ER integrity and dynamics remain unclear. Here, we identify vimentin intermediate filaments as perinuclear anchors for the ER and endolysosomes. We show that perinuclear vimentin filaments engage the ER‐embedded RING finger protein 26 (RNF26) at the C‐terminus of its RING domain. This restricts RNF26 to perinuclear ER subdomains and enables the corresponding spatial retention of endolysosomes through RNF26‐mediated membrane contact sites (MCS). We find that both RNF26 and vimentin are required for the perinuclear coalescence of the ERQC and its juxtaposition with proteolytic compartments, which facilitates efficient recovery from ER stress via the Sec62‐mediated ER‐phagy pathway. Collectively, our findings reveal a scaffolding mechanism that underpins the spatiotemporal integration of organelles during cellular proteostasis.

The endolysosomal system comprises a dynamic constellation of vesicles working together to sense and interpret environmental cues and facilitate homeostasis. Integrating extracellular information with the internal affairs of the cell requires endosomes and lysosomes to be proficient in decision-making: fusion or fission; recycling or degradation; fast transport or contacts with other organelles. To effectively discriminate between these options, the endolysosomal system employs complex regulatory strategies that crucially rely on reversible post-translational modifications (PTMs) with ubiquitin (Ub) and ubiquitin-like (Ubl) proteins. The cycle of conjugation, recognition and removal of different Ub- and Ubl-modified states informs cellular protein stability and behavior at spatial and temporal resolution and is thus well suited to finetune macromolecular complex assembly and function on endolysosomal membranes. Here, we discuss how ubiquitylation (also known as ubiquitination) and its biochemical relatives orchestrate endocytic traffic and designate cargo fate, influence membrane identity transitions and support formation of membrane contact sites (MCSs). Finally, we explore the opportunistic hijacking of Ub and Ubl modification cascades by intracellular bacteria that remodel host trafficking pathways to invade and prosper inside cells.