Christopher L Brett

Concordia University Montreal · Department of Biology

Diverse physiology relies on receptor and transporter protein down-regulation and degradation mediated by ESCRTs. Loss-of-function mutations in human ESCRT genes linked to cancers and neurological disorders are thought to block this process. However, when homologous mutations are introduced into model organisms, cells thrive and degradation persist...

The role of sphingolipids in controlling the endolysosomal membrane trafficking remains unclear. Here, we show that in Saccharomyces cerevisiae sphingolipids containing very long-chain fatty-acids (VLCAs) promote homotypic vacuolar fusion. Yeast that lack the C26 VLCA elongase Elo3p display morphological and vacuolar abnormalities. Vacuoles isolate...

Endocytosis is a fundamental process underlying diverse eukaryotic physiology. The terminal stage of this process is membrane fusion between the perimeter membrane of a late endosome filled with intraluminal vesicles, or multivesicular body (MVB), and the lysosome membrane to facilitate catabolism of internalized biomaterials or surface polytopic p...

SNARE-mediated membrane fusion is required for membrane trafficking as well as organelle biogenesis and homeostasis. The membrane fusion reaction involves sequential formation of hemifusion intermediates, whereby lipid monolayers partially mix on route to complete bilayer merger. Studies of the Saccharomyces cerevisiae lysosomal vacuole have reveal...

Surface receptor and transporter protein down-regulation is assumed to be exclusively mediated by the canonical multivesicular body (MVB) pathway and ESCRTs (Endosomal Sorting Complexes Required for Transport). However, few surface proteins are known to require ESCRTs for down-regulation, and reports of ESCRT-independent degradation are emerging, s...

Upon vacuolar lysosome (or vacuole) fusion in S. cerevisiae, a portion of membrane is internalized and catabolized. Formation of this intralumenal fragment (ILF) is important for organelle protein and lipid homeostasis and remodeling. But how ILF formation is optimized for membrane turnover is not understood. Here, we show that fewer ILFs form when...

Organelle morphology reflects an equilibrium between membrane fusion and fission that determines size, shape and copy number. By studying the yeast vacuole as a model, the conserved molecular mechanisms responsible for organelle fusion have been revealed. However, a detailed understanding of vacuole fission and how these opposing processes respond...

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 lysoso...

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 degrada...

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 l...

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 degrada...

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-indepen...

Lysosomes rely on their resident transporter proteins to return products of catabolism to the cell for reuse and for cellular signaling, metal storage, and maintaining the lumenal environment. Despite their importance, little is known about the lifetime of these transporters or how they are regulated. Using Saccharomyces cerevisiae as a model, we d...

Lysosomal membrane fusion mediates the last step of the autophagy and endocytosis pathways, and supports organelle remodeling and biogenesis. Because fusogenic proteins and lipids concentrate in a ring at the vertex between apposing organelle membranes, the encircled area of membrane can be severed and internalized within the lumen as a fragment up...

Myopathies are a clinically and etiologically heterogeneous group of disorders that can range from limb girdle muscular dystrophy (LGMD) to syndromic forms with associated features including intellectual disability. Here, we report the identification of mutations in transport protein particle complex 11 (TRAPPC11) in three individuals of a consangu...

Self-assembled micelles of amphiphilic block copolymers (ABPs) with stimuli-responsive degradation (SRD) property have a great promise as nanotherapeutics exhibiting enhanced release of encapsulated therapeutics into targeted cells. Here, thiol-responsive degradable micelles based on a new ABP consisting of a pendant disulfide-labeled methacrylate...

Preservation of both the integrity and fluidity of biological membranes is a critical cellular homeostatic function. Signaling pathways that govern lipid bilayer fluidity have long been known in bacteria, yet no such pathways have been identified in eukaryotes. Here we identify mutants of the yeast Saccharomyces cerevisiae whose growth is different...

All four MAP3Ks influence C16:1-sensitivity. SSK2 and/or SSK22 were deleted from the bck1Δ ste11Δ strain using standard methods to give the two triple and the quadruple deletion strains. Growth was at 30° for 3 days. (PDF)

Complete set of growth conditions for rho1 strains, some of which are presented in Figure 4E . (PDF)

Example of data from the screen. Yeast from plate #114 of the MATα deletion collection (one of 52 plates) was applied using a robot to plates containing semi-synthetic medium lacking glucose and containing 1% tergitol, and grown for two weeks at 25°. Plate A lacked fatty acids, plate B contained 0.1% C18:1 and plate C contained 0.1% C16:1. The gene...

pkc1Δ strains readily acquire suppressors of slow growth. Asci from a sporulated PKC1/pkc1Δ strain were dissected on a single plate of YPD medium containing 1 M sorbitol and photographed first at 4 days and then at 14 days of growth at 30°. (PDF)

All four ratios of acyl group content. Total PL from each of four strains was purified in multiple experiments and acyl chain content quantified (Table S2 contains the complete data set). Error bars are the standard error of the mean. (PDF)

Gene deletions which cause oleic acid (OA, C18:1) and/or palmitoleic acid (PO, C16:1) to influence growth. This data is provided as an Excel file. (XLS)

Benzyl Alcohol (BA) and C18:1 counteract the effects of each other on growth. (A) Growth inhibition of a pkc1Δ strain caused by BA is relieved by C18:1. Cells (3, 10-fold serial dilutions) were applied to plates of YPD medium containing 1 M sorbitol and 1% tergitol in the presence (bottom) or absence (top) of BA and in the presence (right) or absen...

The multivesicular body (MVB) is an endosomal intermediate containing intralumenal vesicles destined for membrane protein degradation in the lysosome. In Saccharomyces cerevisiae, the MVB pathway is composed of 17 evolutionarily conserved ESCRT (endosomal sorting complex required for transport) genes grouped by their vacuole protein sorting Class E...

Rab small G proteins control membrane trafficking events required for many processes including secretion, lipid metabolism, antigen presentation and growth factor signaling. Rabs recruit effectors that mediate diverse functions including vesicle tethering and fusion. However, many mechanistic questions about Rab-regulated vesicle tethering are unre...

Growth of yeast mutant strains does not correlate with pHv. Defects in vacuole pH do not correlate with poor growth. Vacuole pH values shown in Figure 1b n (≥4469) are plotted against yeast culture growth measured under acidic (top), standard (middle) or alkaline (bottom panel) conditions. Resulting log-log plots indicate that vacuole pH and growth...

Vesicle trafficking defects lead to pHv dysregulation. Mutants with defective pHv also identified by genome-wide screens for endocytic trafficking defects and/or vacuole fusion defects are shown (numbers indicated in parentheses). Arrows indicate directional trafficking pathways between the following organelles: vacuole (Vac), late endosome (LE), i...

Major functional groups of the vacuolar pH-stat. Genes identified in the vacuolar pH survey of the yeast single gene deletion library were sorted into functional categories. Major functional groups of membrane phospholipid and sterol distribution, endocytic trafficking and transporters are listed. Growth and vacuole pH values obtained from strains...

Summary of major contributors to vacuolar pH-stat. Summary of 107 genes identified as contributors to the vacuole pH-stat under more than one external pH condition (see Figure 1d). The genes are sorted by effect of deletion on vacuole pH and by cellular function. (PDF)

Complete dataset of yeast vacuole pH and growth measured at three external pH values. Vacuole pH and culture growth (Absorbance at 600 nm) values for 4,606 yeast single gene deletion strains examined in this study. Prior to recording, cultures were grown for 19 hours in APG media adjusted to pH 2.7, 4.0 or 7.0. Vacuole pH datasets (also plotted in...

Protons, the smallest and most ubiquitous of ions, are central to physiological processes. Transmembrane proton gradients drive ATP synthesis, metabolite transport, receptor recycling and vesicle trafficking, while compartmental pH controls enzyme function. Despite this fundamental importance, the mechanisms underlying pH homeostasis are not entire...

Traffic through late endolysosomal compartments is regulated by sequential signaling of small G proteins of the Rab5 and Rab7 families. The Saccharomyces cerevisiae Vps-C protein complexes CORVET (class C core vacuole/endosome tethering complex) and HOPS (homotypic fusion and protein transport) interact with endolysosomal Rabs to coordinate their s...

Genetic studies in yeast, plants, insects, and mammals have identified four universally conserved proteins, together called Vps Class C, that are essential for late endosome and lysosome assembly and for numerous endolysosomal trafficking pathways, including the terminal stages of autophagy. Two Vps-C complexes, HOPS and CORVET, incorporate diverse...

Rab guanosine triphosphatases (GTPases) are pivotal regulators of membrane identity and dynamics, but the in vivo pathways that control Rab signaling are poorly defined. Here, we show that the GTPase-activating protein Gyp7 inactivates the yeast vacuole Rab Ypt7 in vivo. To efficiently terminate Ypt7 signaling, Gyp7 requires downstream assistance f...

Osmotic gradients across organelle and plasma membranes modulate the rates of membrane fission and fusion; sufficiently large gradients can cause membrane rupture [1-6]. Hypotonic gradients applied to living yeast cells trigger prompt (within seconds) swelling and fusion of Saccharomyces cerevisiae vacuoles, whereas hypertonic gradients cause vacuo...

In hair cells of the inner ear, robust Ca2+/H+ exchange mediated by plasma-membrane Ca2+-ATPase would rapidly acidify mechanically sensitive hair bundles without efficient removal of H+. We found that, whereas the basolateral membrane of vestibular hair cells from the frog saccule extrudes H+ via an Na+-dependent mechanism, bundles rapidly remove H...

Yeast Nhx1 [Na+(K+)/H+ exchanger 1] is an intracellular Na+(K+)/H+ exchanger, localizing to the late endosome where it is important for ion homoeostasis and vesicle trafficking. Phylogenetic analysis of NHE (Na+/H+ exchanger) sequences has identified orthologous proteins, including HsNHE6 (human NHE6), HsNHE7 and HsNHE9 of unknown physiological rol...

Inherent to the proteome itself, may be information that enables proteins to buffer pH at a level that promotes their own function within a specialized compartment. We observe that the distribution of computed isoelectric points in the yeast proteome matches experimentally derived organellar pH estimates across distinct subcellular compartments. Th...

The intestinal and renal proximal tubule brush border (BB) Na+-H+ exchanger NHE3 binds to members of the NHERF (Na+-H+ exchanger regulatory factor) family. These are four proteins (current most used names include NHERF1, NHERF2, PDZK1 and IKEPP) which are related to each other, are present in locations in or close to the BB, and scaffold a variable...

The relationship between endosomal pH and function is well documented in viral entry, endosomal maturation, receptor recycling, and vesicle targeting within the endocytic pathway. However, specific molecular mechanisms that either sense or regulate luminal pH to mediate these processes have not been identified. Herein we describe the use of novel,...

More than 200 genes annotated as Na+/H+ hydrogen exchangers (NHEs) currently reside in bioinformation databases such as GenBank and Pfam. We performed detailed phylogenetic analyses of these NHEs in an effort to better understand their specific functions and physiological roles. This analysis initially required examining the entire monovalent catio...

• We examined the effects of noradrenaline on steady-state intracellular pH (pHi) and the recovery of pHi from internal acid loads imposed by the NH4+ prepulse technique in hippocampal CA1 neurones acutely dissociated from adult rats. • Under nominally HCO3−-free conditions, acid extrusion was accomplished by a Na+-dependent mechanism, probably the...

Endosomal Na+/H+ exchangers are important for salt and osmotolerance, vacuolar pH regulation, and endosomal trafficking. We show that the C terminus of yeast Nhx1 interacts with Gyp6, a GTPase-activating protein for the Ypt/Rab family of GTPases, and that Gyp6 colocalizes with Nhx1 in the endosomal/prevacuolar compartment (PVC). The gyp6 null mutan...

The contributions of HCO(3)(-)-dependent, DIDS-sensitive mechanisms to the maintenance of steady-state pH(i), and the regulation of their activities by cAMP-dependent protein kinase (PKA), were investigated in CA1 neurons with the H(+)-sensitive fluorophore, BCECF. The addition of HCO(3)(-)/CO(2) to neurons with "low" (pH(i) < or = 7.20) and "high"...

Since the discovery of the first intracellular Na(+)/H(+) exchanger in yeast, Nhx1, multiple homologs have been cloned and characterized in plants. Together, studies in these organisms demonstrate that Nhx1 is located in the prevacuolar/vacuolar compartment of cells where it sequesters Na(+) into the vacuole, regulates intravesicular pH, and contri...

The ratiometric indicators 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein and Fura-2 were employed to examine, respectively, intracellular pH (pHi) and calcium ([Ca²⁺]i) changes evoked by anoxia in cultured postnatal rat hippocampal neurons at 37°C. Under both HCO⁻3/CO2- and HEPES-buffered conditions, 3-, 5- or 10-min anoxia induced a trip...

On title page, first word of title appears as "pHOME"; in Abstract [p. ii], it is referred to as "pHome". Vita. U.M.I. no. 3172553. Thesis (Ph. D.)--Johns Hopkins University, 2005. Includes bibliographical references. Microfilm.