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Re-localization of components of the guided entry of tail-anchored proteins targeting pathway to cytosolic foci upon glucose starvation

ABSTRACT: Protein targeting is an essential step in the biogenesis of membrane proteins and the guided-entry of tail-anchored proteins (GET) pathway is a key targeting route to the endoplasmic reticulum. The adaptive response to cellular stress is multifaceted but a hallmark is the formation of membraneless organelles, which can act as sequestration sites for particular factors, allowing regulation of cellular processes during stress and/or facilitating efficient recovery upon stress alleviation. Upon glucose withdrawal, we observe that the Get3 ATPase of the GET pathway re-locates to dynamic cytosolic foci, here termed GET bodies, that also contain proteins of the GET pathway pre-targeting complex. Our data demonstrate that Get3 and the Get4-Get5 heterodimer play important roles in the assembly of these structures, and we further uncover the requirement of the TPR domain of Sgt2 for the GET body formation. Compositional analyses of GET bodies by enrichment coupled to mass spectrometry as well as high-throughput microscopy-based screening reveal a chaperone-rich core and the association of an inventory of proteins involved in metabolic processes. Temporal analyses of GET body assembly demonstrate the sequential recruitment of different chaperones and we discover the requirement of the co-chaperones Sis1 and Sti1 for maintaining the dynamic properties of these structures. As lack of Adh2, which converts ethanol to acetaldehyde in a reaction coupled to NADH production, impairs GET body disassembly, and the addition of NADH promotes GET body dissolution in vitro, our results suggest a mechanistic basis for the formation of GET bodies in response to the metabolic shift caused by glucose withdrawal.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Christof Lenz

LAB HEAD: Christof Lenz

PROVIDER: PXD054296 | Pride | 2025-02-25

REPOSITORIES: pride

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			Action	DRS
	J_Jennich_060422_140422_HFGoe_L1_rep1_1.raw	Raw
	J_Jennich_060422_140422_HFGoe_L1_rep1_10.raw	Raw
	J_Jennich_060422_140422_HFGoe_L1_rep1_11.raw	Raw
	J_Jennich_060422_140422_HFGoe_L1_rep1_12.raw	Raw
	J_Jennich_060422_140422_HFGoe_L1_rep1_13.raw	Raw

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Project description:The subcellular localization of specific mRNAs is an evolutionary conserved mechanism that underlies the establishment of cellular polarity and specialized cell functions. In neurons, mRNA trafficking and local protein translation in dendrites provides an important mechanism that mediates synaptic development and plasticity. The significance of mRNA targeting and protein synthesis in axons, however, is still unclear. Only a small number of transcripts have been identified in axons to date, and their contribution to axon growth and neuronal survival remains largely unknown. Here, we report the results of a novel screen that allowed the separate identification of mRNAs localized in cell bodies and in axons of developing neurons. Using compartmentalized cultures of sympathetic neurons and Sequential Analysis of Gene Expression (SAGE), the screen identified more than 200 axonal mRNAs, including ones that encode cytoskeletal proteins and proteins that function in neural development and signal transduction. Importantly, several classes of transcripts were selectively enriched in axons, indicating that an active process drives the targeting of specific mRNAs from the cell bodies to the axons. This study is the first comprehensive and unbiased analysis of mRNA localization in subcellular domains of any neuronal cell type. We used compartmentalized chambers to culture neonatal rat sympathetic neurons (Campenot, 1977). In these cultures, the cell bodies are separated from the distal axons by a 1 mm wide Teflon divider, which maintains the cell bodies and axon terminals in separate fluid compartments. Primary rat sympathetic neurons are especially suitable for compartmentalized culture because they can be grown as a highly homogeneous population without glial cells. Neurons were seeded in the central compartment with nerve growth factor (NGF), and after a few days, the NGF was lowered in this compartment and supplied only to the peripheral compartment to stimulate axon growth. The anti-mitotic agent cytosine arabinoside C (Ara-C) was added to both compartments to remove non-neuronal cells. mRNA was then isolated after 12 days in culture (DIV) from cell body or axon compartments. As the initial mRNA content in axons was not sufficient to perform the SAGE analysis, both axon and cell body mRNAs were subjected to two rounds of linear amplification to obtain antisense RNA (aRNA). The amplified aRNA was then reverse transcribed and second strand synthesis was performed to proceed with the SAGE assay using the LongSAGE kit (Invitrogen) according to the manufacturer’s protocol.

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Re-localization of components of the guided entry of tail-anchored proteins targeting pathway to cytosolic foci upon glucose starvation

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