Dataset Information

Functional characterization of 67 endocytic accessory proteins using multiparametric quantitative analysis of CCP dynamics.

ABSTRACT: Clathrin-mediated endocytosis (CME) begins with the nucleation of clathrin assembly on the plasma membrane, followed by stabilization and growth/maturation of clathrin-coated pits (CCPs) that eventually pinch off and internalize as clathrin-coated vesicles. This highly regulated process involves a myriad of endocytic accessory proteins (EAPs), many of which are multidomain proteins that encode a wide range of biochemical activities. Although domain-specific activities of EAPs have been extensively studied, their precise stage-specific functions have been identified in only a few cases. Using single-guide RNA (sgRNA)/dCas9 and small interfering RNA (siRNA)-mediated protein knockdown, combined with an image-based analysis pipeline, we have determined the phenotypic signature of 67 EAPs throughout the maturation process of CCPs. Based on these data, we show that EAPs can be partitioned into phenotypic clusters, which differentially affect CCP maturation and dynamics. Importantly, these clusters do not correlate with functional modules based on biochemical activities. Furthermore, we discover a critical role for SNARE proteins and their adaptors during early stages of CCP nucleation and stabilization and highlight the importance of GAK throughout CCP maturation that is consistent with GAK's multifunctional domain architecture. Together, these findings provide systematic, mechanistic insights into the plasticity and robustness of CME.

SUBMITTER: Bhave M

PROVIDER: S-EPMC7749282 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Functional characterization of 67 endocytic accessory proteins using multiparametric quantitative analysis of CCP dynamics.

Bhave Madhura M Mino Rosa E RE Wang Xinxin X Lee Jeon J Grossman Heather M HM Lakoduk Ashley M AM Danuser Gaudenz G Schmid Sandra L SL Mettlen Marcel M

Proceedings of the National Academy of Sciences of the United States of America 20201130 50

Clathrin-mediated endocytosis (CME) begins with the nucleation of clathrin assembly on the plasma membrane, followed by stabilization and growth/maturation of clathrin-coated pits (CCPs) that eventually pinch off and internalize as clathrin-coated vesicles. This highly regulated process involves a myriad of endocytic accessory proteins (EAPs), many of which are multidomain proteins that encode a wide range of biochemical activities. Although domain-specific activities of EAPs have been extensive ...[more]

PMID: 33257546

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Project description:In the apicomplexans, endocytosed cargos (e.g., hemoglobin) are trafficked to a specialized organelle for digestion. This follows a unique endocytotic process at the micropore/cytostome in these parasites. However, the mechanism underlying endocytic trafficking remains elusive, due to the repurposing of classical endocytic proteins for the biogenesis of apical organelles. To resolve this issue, we have exploited the genetic tractability of the model apicomplexan Toxoplasma gondii, which ingests host cytosolic materials (e.g., green fluorescent protein[GFP]). We determined an association between protein prenylation and endocytic trafficking, and using an alkyne-labeled click chemistry approach, the prenylated proteome was characterized. Genome editing, using clustered regularly interspaced short palindromic repaet/CRISPR-associated nuclease 9 (CRISPR/Cas9), was efficiently utilized to generate genetically modified lines for the functional screening of 23 prenylated candidates. This identified four of these proteins that regulate the trafficking of endocytosed GFP vesicles. Among these proteins, Rab1B and YKT6.1 are highly conserved but are non-classical endocytic proteins in eukaryotes. Confocal imaging analysis showed that Rab1B and Ras are substantially localized to both the trans-Golgi network and the endosome-like compartments in the parasite. Conditional knockdown of Rab1B caused a rapid defect in secretory trafficking to the rhoptry bulb, suggesting a trafficking intersection role for the key regulator Rab1B. Further experiments confirmed a critical role for protein prenylation in regulating the stability/activity of these proteins (i.e., Rab1B and YKT6.1) in the parasite. Our findings define the molecular basis of endocytic trafficking and reveal a potential intersection function of Rab1B on membrane trafficking in T. gondii. This might extend to other related protists, including the malarial parasites. IMPORTANCE The protozoan Toxoplasma gondii establishes a permissive niche, in host cells, that allows parasites to acquire large molecules such as proteins. Numerous studies have demonstrated that the parasite repurposes the classical endocytic components for secretory sorting to the apical organelles, leaving the question of endocytic transport to the lysosome-like compartment unclear. Recent studies indicated that endocytic trafficking is likely to associate with protein prenylation in malarial parasites. This information promoted us to examine this association in the model apicomplexan T. gondii and to identify the key components of the prenylated proteome that are involved. By exploiting the genetic tractability of T. gondii and a host GFP acquisition assay, we reveal four non-classical endocytic proteins that regulate the transport of endocytosed cargos (e.g., GFP) in T. gondii. Thus, we extend the principle that protein prenylation regulates endocytic trafficking and elucidate the process of non-classical endocytosis in T. gondii and potentially in other related protists.

Dataset Information

Functional characterization of 67 endocytic accessory proteins using multiparametric quantitative analysis of CCP dynamics.

Publications

Functional characterization of 67 endocytic accessory proteins using multiparametric quantitative analysis of CCP dynamics.

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