Biology subjects

Brett, C.

Publications and source records attributed to Brett, C..

4 recordsLinked to original sources

bioRxiv2017

ILF and vReD pathways cooperate to control lysosomal transporter protein lifetimes

Lysosomal nutrient transporter proteins move lumenal products of biomaterial catabolism to the cytoplasm for reuse by the cell. Two mechanisms control their lifetimes: the ILF (IntraLumenal Fragment) and vReD (Vacuole REcycling and Degradation) pathways. But it is not clear if they function independently. Using S. cerevisiae as a model, here we show that the ILF pathway mediates constitutive turnover of the lysine transporter Ypq1 and zinc transporter Cot1--known vReD client proteins--in vivo and in vitro. In contrast, the vReD pathway mediates constitutive degradation of the amino acid transporter Vba4. Activation of TOR with cycloheximide enhances their degradation by these pathways. However, misfolding by heat stress shunts all three into the ILF pathway. Thus, both pathways control individual transporter lifetimes, although only the ILF pathway mediates protein quality control. The pathway chosen depends on protein fate: degradation is imminent by the ILF pathway, whereas the vReD pathway permits reuse.\n\nHIGHLIGHTS and eTOC BLURBO_LIvReD and ILF pathways control Ypq1, Cot1 and Vba4 lifetimes\nC_LIO_LIILF pathway constitutively degrades Ypq1 and Cot1, vReD degrades Vba4\nC_LIO_LITOR activation stimulates protein degradation by both pathways\nC_LIO_LIILF pathway clears all misfolded proteins for quality control\nC_LI\n\nTwo mechanisms degrade lysosome transporter proteins but it is not clear how each contributes to their lifetimes for organelle homeostasis or remodeling. Here McNally and Brett show that the transporters Cot1, Ypq1 and Vba4 can be selectively degraded by the ILF (IntraLumenal Fragment) pathway or vReD (Vacuole REcycling and Degradation) pathway depending on stimulus. However, only the ILF pathway mediates protein quality control.

cell biology↗

bioRxiv2017

The Na+(K+)/H+ exchanger Nhx1 controls multivesicular body-vacuolar lysosome fusion

Loss-of-function mutations in human endosomal Na+(K+)/H+ Exchangers (NHEs) NHE6 and NHE9 are implicated in neurological disorders including Christianson Syndrome, autism and attention deficit and hyperactivity disorder (ADHD). These mutations disrupt retention of surface receptors within neurons and glial cells by affecting their delivery to lysosomes for degradation. However, the molecular basis of how these endosomal NHEs control endocytic trafficking is unclear. Using Saccharomyces cerevisiae as a model, we conducted cell-free organelle fusion assays to show that transport activity of the orthologous endosomal NHE Nhx1 is important for multivesicular body (MVB)-vacuolar lysosome fusion, the last step of endocytosis required for surface protein degradation. We find that deleting Nhx1 disrupts the fusogenicity of the MVB, not vacuole, by targeting pH-sensitive machinery downstream of the Rab-GTPase Ypt7 needed for SNARE-mediated lipid bilayer merger. All contributing mechanisms are evolutionarily conserved offering new insight into the etiology of human disorders linked to loss of endosomal NHE function.

cell biology↗

bioRxiv2017

ESCRT-Independent Surface Receptor And Transporter Protein Degradation By The ILF Pathway

Surface receptor and transporter protein down-regulation drives cell signaling, quality control and metabolism underlying diverse physiology. After endocytosis, proteins are delivered to endosomes where ESCRTs package them into intralumenal vesicles, which are degraded by acid hydrolases upon fusion with lysosomes. However, reports of ESCRT-independent surface protein degradation are emerging suggesting that alternative, non-canonical pathways exist. Using Saccharomyces cerevisiae as a model, here we show that in response to substrates, protein misfolding or TOR signaling, some internalized surface transporters (Hxt3, Itr1, Aqr1) bypass ESCRTs en route to the lysosome membrane where they are sorted into an area that is internalized as an intralumenal fragment (ILF) and degraded upon organelle fusion. This ILF pathway also degrades typical ESCRT client proteins (Mup1, Can1, Ste3) when ESCRT function is impaired. As the underlying machinery is conserved, we speculate that the ILF pathway is an important contributor to receptor and transporter down-regulation in all eukaryotes.

cell biology↗

bioRxiv2017

Distinct features of multivesicular body-lysosome fusion revealed by a new cell-free content-mixing assay

When marked for degradation, surface receptor and transporter proteins are internalized and delivered to endosomes where they are packaged into intralumenal vesicles (ILVs). Many rounds of ILV formation create multivesicular bodies (MVBs) that fuse with lysosomes exposing ILVs to hydrolases for catabolism. Despite being critical for protein degradation, the molecular underpinnings of MVB-lysosome fusion remain unclear, although machinery underlying other lysosome fusion events is implicated. But how then is specificity conferred? And how is MVB maturation and fusion coordinated for efficient protein degradation? To address these questions, we developed a cell-free MVB-lysosome fusion assay using S. cerevisiae as a model. After confirming that the Rab7 ortholog Ypt7 and the multisubunit tethering complex HOPS are required, we found that the Qa-SNARE Pep12 distinguishes this event from homotypic lysosome fusion. Mutations that impair MVB maturation block fusion by preventing Ypt7 activation, confirming that a Rab-cascade mechanism harmonizes MVB maturation with lysosome fusion.\n\nIMPACT STATEMENTEndocytosis culminates with multivesicular bodies (MVBs) fusing with lysosomes. But the molecular underpinnings of this event remain unclear. Here, using S. cerevisiae as a model, Karim et al. employ a new in vitro assay to show that MVB-lysosome fusion is driven by ESCRT-dependent Rab-GTPase activation and the syntaxin ortholog Pep12, distinguishing it from other lysosome membrane fusion events.

cell biology↗