Biology subjects

Pashkova, N.

Publications and source records attributed to Pashkova, N..

4 recordsLinked to original sources

bioRxiv2025

Cargo adaptors use a handhold mechanism to engage with myosin V for organelle transport

Myo2, a myosin V motor, is essential for organelle transport in budding yeast. Its attachment to and detachment from cargo are mediated by adaptor molecules. Vac17, a vacuole-specific adaptor, links Myo2 to Vac8 on the vacuole membrane, and plays a key role in the formation and dissociation of the Myo2-Vac17-Vac8 complex. Using genetics, cryo-electron microscopy and structure prediction, we find that Vac17 interacts with Myo2 through two distinct sites rather than a single interface. Similarly, the peroxisome adapter Inp2 engages two separate regions of Myo2, one of which overlaps with Vac17. These findings support a "handhold" model, in which cargo adaptors occupy multiple sites on the Myo2 tail, enhancing motor-cargo interactions and likely providing additional regulatory control over motor recruitment. SummaryThis study provides insights into how cargo adaptors bind myosin V. Genetics, cell-based assays, cryo-EM, and AlphaFold, reveal that the vacuole-specific adaptor uses a handhold mechanism to attach to two areas on the myosin V tail. Moreover, evidence is presented that other adaptors use a similar strategy.

cell biology↗

bioRxiv2023

PMP22 associates with MPZ via their transmembrane domains and disrupting this interaction causes a loss-of-function phenotype similar to hereditary neuropathy associated with liability to pressure palsies (HNPP).

PMP22 and MPZ are abundant myelin membrane proteins in Schwann cells. The MPZ adhesion protein holds myelin wraps together across the intraperiod line. PMP22 is a tetraspan protein belonging to the Claudin superfamily. Loss of either MPZ or PMP22 causes severe demyelinating Charcot-Marie-Tooth (CMT) peripheral neuropathy, and duplication of PMP22 causes the most common form of CMT, CMT1A. Yet, the molecular functions provided by PMP22 and how its alteration causes CMT are unknown. Here we find MPZ and PMP22 form a specific complex through interfaces within their transmembrane domains. We also find that the PMP22 A67T patient variant that causes a loss-of-function (Hereditary Neuropathy with Pressure Palsies) phenotype maps to this interface, and blocks MPZ association without affecting localization to the plasma membrane or interactions with other proteins. These data define the molecular basis for the MPZ~PMP22 interaction and indicate this complex fulfills an important function in myelinating cells.

neuroscience↗

bioRxiv2021

ANTH domains within CALM, HIP1R, and Sla2 recognize ubiquitin internalization signals.

Ubiquitin (Ub) serves as a signal for clathrin-mediated endocytosis (CME) by engaging Ub-binding proteins with the internalization apparatus. Ub is a versatile internalization signal because it can be added to a wide variety of membrane proteins, expanding the capacity of cells to use a variety of regulatory mechanisms to specify the conditions under which a particular protein will be internalized. Several candidate adaptors that can recognize ubiquitinated membrane proteins have been identified that work in endocytic processes that are both clathrin-dependent and independent. These include Epsin and Eps15, which bind and help sort Ub-cargo into internalization sites. Here we identify additional components of the endocytosis apparatus that bind Ub. The N-terminal ANTH domains found in CALM, AP180, HIP1R and yeast Sla2 all bind monoubiquitin with {micro}M affinity. ANTH domains belong to a larger superfamily of domains including ENTH and VHS domains, many of which have Ub-binding regions outside of their VHS/ENTH/ANTH domains that enable them to mediate Ub-dependent sorting events throughout the cell. Solution NMR studies combined with a crystal structure of the CALM ANTH domain in a complex with Ub show that Ub binds to a C-terminal region of the ANTH domain that is not present in ENTH domains. Combined loss of Ub-binding by ANTH-domain proteins and other Ub-binding domains within the internalization apparatus of yeast caused defects in the Ub-dependent internalization of the GPCR Ste2 but had no effect on internalization of Ste2 via other internalization signals. These studies define new components of the internalization machinery that work collectively with Epsin and Eps15 to specify recognition of Ub as an internalization signal.

cell biology↗

bioRxiv2020

Bro1 directly stimulates Vps4 activity to promote Intralumenal Vesicle Formation during Multivesicular Body biogenesis

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homolog of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT-driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologs in licensing ESCRT-III-dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1/Vps4/ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition. SummaryCargo sorting is coordinated with intralumenal vesicle budding during ESCRT-mediated multivesicular body biogenesis. Bro1 V domain stimulates Vps4 to promote ESCRT-III-driven intralumenal vesicle formation in a manner required for this coordinated process.

cell biology↗