VMA21 deficiency causes an autophagic myopathy by compromising V-ATPase activity and lysosomal acidification.

X-linked myopathy with excessive autophagy (XMEA) is a childhood-onset disease characterized by progressive vacuolation and atrophy of skeletal muscle. We show that XMEA is caused by hypomorphic alleles of the VMA21 gene, that VMA21 is the diverged human ortholog of the yeast Vma21p protein, and that like Vma21p it is an essential assembly chaperone of the V-ATPase, the principal mammalian proton pump complex. Decreased VMA21 raises lysosomal pH, which reduces lysosomal degradative ability and blocks autophagy. This reduces cellular free amino acids, which upregulates the mTOR pathway and mTOR-dependent macroautophagy, resulting in proliferation of large and ineffective autolysosomes that engulf sections of cytoplasm, merge together, and vacuolate the cell. Our results uncover macroautophagic overcompensation leading to cell vacuolation and tissue atrophy as a mechanism of disease.

VMA21 deficiency prevents vacuolar ATPase assembly and causes autophagic vacuolar myopathy.

X-linked Myopathy with Excessive Autophagy (XMEA) is a childhood onset disease characterized by progressive vacuolation and atrophy of skeletal muscle. We show that XMEA is caused by hypomorphic alleles of the VMA21 gene, that VMA21 is the diverged human ortholog of the yeast Vma21p protein, and that like Vma21p, VMA21 is an essential assembly chaperone of the vacuolar ATPase (V-ATPase), the principal mammalian proton pump complex. Decreased VMA21 raises lysosomal pH which reduces lysosomal degradative ability and blocks autophagy. This reduces cellular free amino acids which leads to downregulation of the mTORC1 pathway, and consequent increased macroautophagy resulting in proliferation of large and ineffective autolysosomes that engulf sections of cytoplasm, merge, and vacuolate the cell. Our results uncover a novel mechanism of disease, namely macroautophagic overcompensation leading to cell vacuolation and tissue atrophy.

Lipid metabolism and dynamics during phagocytosis.

Phagocytosis, the engulfment of particles, mediates the elimination of invading pathogens as well as the clearance of apoptotic cells. Ingested particles reside within a vacuole or phagosome, where they are eventually destroyed and digested. The phagosomal lumen acquires microbicidal and digestive properties through interaction with various components of the endocytic pathway, a process known as maturation. Lipids are known to have numerous roles in phagosome formation and maturation; recent developments in the design of lipid-specific probes and in high-resolution imaging have revealed that lipids, notably phosphoinositides, are involved in signaling, actin assembly and the recruitment of molecular motors to sites of ingestion. In addition, phosphoinositides and other lipids also regulate multiple membrane budding, fission and fusion events required for maturation.

Differential intracellular transport and binding of verotoxin 1 and verotoxin 2 to globotriaosylceramide-containing lipid assemblies.

Although verotoxin-1 (VT1) and verotoxin-2 (VT2) share a common receptor, globotriaosyl ceramide (Gb(3)), VT2 induces distinct animal pathology and is preferentially associated with human disease. Moreover VT2 cytotoxicity in vitro is less than VT1. We therefore investigated whether these toxins similarly traffic within cells via similar Gb(3) assemblies. At 4 degrees C, fluorescent-VT1 and VT2 bound both coincident and distinct punctate surface Gb(3) microdomains. After 10 min at 37 degrees C, similar distinct/coincident micropunctate intracellular localization was observed. Most internalized VT2, but not VT1, colocalized with transferrin. After 1 h, VT1 and VT2 coalesced during retrograde transport to the Golgi. During prolonged incubation (3-6 h), VT1, and VT2 (more slowly), exited the Golgi to reach the ER/nuclear envelope. At this time, VT2 induced a previously unreported, retrograde transport-dependent vacuolation. Cell surface and intracellular VT1 showed greater detergent resistance than VT2, suggesting differential 'raft' association. >90% (125)I-VT1 cell surface bound, or added to detergent-resistant cell membrane extracts (DRM), was in the Gb(3)-containing sucrose gradient 'insoluble' fraction, whereas only 30% (125)I-VT2 was similarly DRM-associated. VT1 bound more efficiently to Gb(3)/cholesterol DRMs generated in vitro. Only VT1 binding was inhibited by high cholesterol/Gb(3) ratios. VT2 competed less effectively for (125)I-VT1/Gb(3) DRM-binding but only VT2-Gb(3)/cholesterol DRM-binding was augmented by sphingomyelin. Differential VT1/VT2 Gb(3) raft-binding may mediate differential cell binding/intracellular trafficking and cytopathology.

The nature of the phagosomal membrane: endoplasmic reticulum versus plasmalemma.

For decades, the vacuole that surrounds particles engulfed by phagocytosis was believed to originate from the plasma membrane. Conversion of the nascent phagosome into a microbicidal organelle was thought to result from the subsequent, orderly fusion of early endosomes, late endosomes, and ultimately, lysosomes with the original plasma membrane-derived vacuole. This conventional model has been challenged, if not superseded, by a revolutionary model that regards phagosome formation as resulting from the particle sliding into the endoplasmic reticulum via an opening at the base of the phagocytic cup. The merits and implications of these two hypotheses are summarized here and analyzed in light of recent results.

Quantitative and dynamic assessment of the contribution of the ER to phagosome formation.

Phagosomes were traditionally thought to originate from an invagination and scission of the plasma membrane to form a distinct intracellular vacuole. An alternative model implicating the endoplasmic reticulum (ER) as a major component of nascent and maturing phagosomes was recently proposed (Gagnon et al., 2002). To reconcile these seemingly disparate hypotheses, we used a combination of biochemical, fluorescence imaging, and electron microscopy techniques to quantitatively and dynamically assess the contribution of the plasmalemma and of the ER to phagosome formation and maturation. We could not verify even a transient physical continuity between the ER and the plasma membrane, nor were we able to detect a significant contribution of the ER to forming or maturing phagosomes in either macrophages or dendritic cells. Instead, our data indicate that the plasma membrane is the main constituent of nascent and newly formed phagosomes, which are progressively remodeled by fusion with endosomal and eventually lysosomal compartments as phagosomes mature into acidic, degradative organelles.

The pH of the secretory pathway: measurement, determinants, and regulation.

The luminal pH of the secretory pathway plays a critical role in the posttranslational modification and sorting of proteins and lipids. The pH of each one of the organelles that constitute the pathway is unique, becoming more acidic as the biosynthetic cargo approaches its destination. The methods used for measurement of pH in the secretory pathway, its determinants, and its regulation are the subjects of this review.

Disruption of a salt bridge dramatically accelerates subunit exchange in duck delta2 crystallin.

Intragenic complementation is a unique property of oligomeric enzymes with which to study subunit-subunit interactions. Complementation occurs when different subunits, each possessing distinct mutations that render the individual homomutant proteins inactive, interact to form a heteromutant protein with partial recovery of activity. In this paper, complementation events between human argininosuccinate lyase (ASL) and its homolog, duck delta2 crystallin, were characterized. Different active site mutants in delta2 crystallin complement by the regeneration of native-like active sites as reported previously for ASL. The complementarity of the ASL and delta2 crystallin subunit interfaces was illustrated by the in vivo formation of active hybrid tetramers from inactive ASL and inactive delta2 crystallin mutants. Subunits of both ASL and delta2 crystallin do not dissociate and reassociate in vitro at room temperature, even after 6 days of incubation, indicating that the multimerization interface is very strong. However, disruption of a salt bridge network in the tetrameric interface of delta2 crystallin caused a drastic acceleration of subunit dissociation. Double mutants combining these interface mutants with active site mutants of delta2 crystallin were able to dissociate and reassociate to form active tetramers in vitro within hours. These results suggest that exchange of subunits may occur without unfolding of the monomer. Intragenic complementation in these interface mutants occurs by reintroducing the native salt bridge interaction upon hetero-oligomerization. Our studies demonstrate the value of intragenic complementation as a tool for investigating subunit-subunit interactions in oligomeric proteins.

Molecular and cellular mechanisms underlying iron transport deficiency in microcytic anemia.

A mutation of the iron transporter Nramp2 (DMT1, Slc11a2) causes microcytic anemia in mk mice and in Belgrade rats by impairing iron absorption in the duodenum and in erythroid cells, causing severe iron deficiency. Both mk and Belgrade animals display a glycine-to-arginine substitution at position 185 (G185R) in the fourth predicted transmembrane domain of Nramp2. To study the molecular basis for the loss of function of Nramp2(G185R), we established cell lines stably expressing extracellularly tagged versions of wild-type (WT) or mutated transporters. Like WT Nramp2, the G185R mutant was able to reach the plasmalemma and endosomal compartments, but with reduced efficiency. Instead, a large fraction of Nramp2(G185R) was detected in the endoplasmic reticulum, where it was unstable and was rapidly degraded by a proteasome-dependent mechanism. Moreover, the stability of the mutant protein that reached the plasma membrane was greatly reduced, further diminishing its surface density at steady state. Last, the specific metal transport activity of plasmalemmal Nramp2(G185R) was found to be significantly depressed, compared with its WT counterpart. Thus, a singlepoint mutation results in multiple biosynthetic and functional defects that combine to produce the impaired iron deficiency that results in microcytic anemia.