GTP Hydrolysis Is Not Important for Ypt1 GTPase Function in Vesicular Transport

GTPases of the Ypt/Rab family play a key role in the regulation of vesicular transport. Their ability to cycle between the GTP- and the GDP-bound forms is thought to be crucial for their function. Conversion from the GTP- to the GDP-bound form is achieved by a weak endogenous GTPase activity, which can be stimulated by a GTPase-activating protein (GAP). Current models suggest that GTP hydrolysis and GAP activity are essential for vesicle fusion with the acceptor compartment or for timing membrane fusion. To test this idea, we inactivated the GTPase activity of Ypt1p by using the Q67L mutation, which targets a conserved residue that helps catalyze GTP hydrolysis in Ras. We demonstrate that the mutant Ypt1-Q67L protein is severely impaired in its ability to hydrolyze GTP both in the absence and in the presence of GAP and consequently is restricted mostly to the GTP-bound form. Surprisingly, a strain with ypt1-Q67L as the only YPT1 gene in the cell has no observable growth phenotypes at temperatures ranging from 14 to 37°C. In addition, these mutant cells exhibit normal rates of secretion and normal membrane morphology as determined by electron microscopy. Furthermore, the ypt1-Q67L allele does not exhibit dominant phenotypes in cell growth and secretion when overexpressed. Together, these results lead us to suggest that, contrary to current models for Ypt/Rab function, GTP hydrolysis is not essential either for Ypt1p-mediated vesicular transport or as a timer to turn off Ypt1p-mediated membrane fusion but only for recycling of Ypt1p between compartments. Finally, the ypt1-Q67L allele, like the wild type, is inhibited by dominant nucleotide-free YPT1 mutations. Such mutations are thought to exert their dominant phenotype by sequestration of the guanine nucleotide exchange factor (GNEF). These results suggest that the function of Ypt1p in vesicular transport requires not only the GTP-bound form of the protein but also the interaction of Ypt1p with its GNEF.     

The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery

A yeast GTP-binding protein, the YPT1 gene product, has been found to function early in the secretion pathway. The ypt1-1 mutation causes a phenotype reminiscent of early secretion-defective mutants, including accumulation of membranes and vesicles as well as a partial defect in secretion and incomplete glycosylation of invertase. Immunofluorescence localization studies using affinity-purified antibody directed against the YPT1 protein showed punctate staining of the cytoplasm of growing yeast cells and very intense staining of small buds, where membrane growth and secretion are most active. The punctate cytoplasmic staining is changed in a mutant (sec7) under conditions that cause aberrant Golgi structures to accumulate. The pattern of immunofluorescence obtained when mouse cells were stained with the antibody coincided closely with the pattern observed with wheat germ agglutinin, suggesting that a mammalian counterpart of the yeast YPT1 protein is located in the Golgi apparatus. These results are interpreted as suggesting that GTP-binding proteins may act to direct intracellular vesicle traffic.     

NEUROCHEMICAL ASPECTS OF NEURONAL ONTOGENESIS IN THE DEVELOPING RAT CEREBELLUM: CHANGES IN NEUROTRANSMITTER AND POLYAMINE SYNTHESIZING ENZYMES

Abstract— Changes in the activities of several specific enzymes were measured in the cerebellum during development. Early transient increases were found in both ornithine decarboxylase and S -adenosylmethionine decarboxylase, enzymes involved in the initial steps of polyamine synthesis. Different patterns of changes were found in neurotransmitter synthesizing enzymes. Tyrosine hydroxylase activity achieved adult levels very early, by 3 days after birth, and remained at this level. Glutamic acid decarboxylase activity, while very low at early stages, increased rapidly before birth and then after a lag period of 10 days started to increase rapidly, directly related to the general growth of cerebellar weight and protein content. Choline acetyltransferase activity started to increase rapidly, reaching a peak of about 100% of adult levels at 3-7 days after birth; the activity then gradually declined and at 20 days, after reaching a low of about 55% of adult values, gradually started to increase, reaching adult levels later than 40 days after birth. The development of protein carboxymethylase activity was similar to that of glutamic acid decarboxylase, directly related to the general growth of the cerebellum. Several interpretations of the results are discussed.     

Effect of geometrical irregularities on propagation delay in axonal trees.

Multiple successive geometrical inhomogeneities, such as extensive arborization and terminal varicosities, are usual characteristics of axons. Near such regions the velocity of the action potential (AP) changes. This study uses AXONTREE, a modeling tool developed in the companion paper for two purposes: (a) to gain insights into the consequence of these irregularities for the propagation delay along axons, and (b) to simulate the propagation of APs along a reconstructed axon from a cortical cell, taking into account information concerning the distribution of boutons (release sites) along such axons to estimate the distribution of arrival times of APs to the axons release sites. We used Hodgkin and Huxley (1952) like membrane properties at 20 degrees C. Focusing on the propagation delay which results from geometrical changes along the axon (and not from the actual diameters or length of the axon), the main results are: (a) the propagation delay at a region of a single geometrical change (a step change in axon diameter or a branch point) is in the order of a few tenths of a millisecond. This delay critically depends on the kinetics and the density of the excitable channels; (b) as a general rule, the lag imposed on the AP propagation at a region with a geometrical ratio GR greater than 1 is larger than the lead obtained at a region with a reciprocal of that GR value; (c) when the electronic distance between two successive geometrical changes (Xdis) is small, the delay is not the sum of the individual delays at each geometrical change, when isolated. When both geometrical changes are with GR greater than 1 or both with GR less than 1, this delay is supralinear (larger than the sum of individual delays). The two other combinations yield a sublinear delay; and (d) in a varicose axon, where the diameter changes frequently from thin to thick and back to thin, the propagation velocity may be slower than the velocity along a uniform axon with the thin diameter. Finally, we computed propagation delays along a morphologically characterized axon from layer V of the somatosensory cortex of the cat. This axon projects mainly to area 4 but also sends collaterals to areas 3b and 3a. The model predicts that, for this axon, areas 3a, 3b, and the proximal part of area 4 are activated approximately 2 ms before the activation of the distal part of area 4.     

Hormone‐induced mitochondrial fission is utilized by brown adipocytes as an amplification pathway for energy expenditure

Adrenergic stimulation of brown adipocytes (BA) induces mitochondrial uncoupling, thereby increasing energy expenditure by shifting nutrient oxidation towards thermogenesis. Here we describe that mitochondrial dynamics is a physiological regulator of adrenergically‐induced changes in energy expenditure. The sympathetic neurotransmitter Norepinephrine (NE) induced complete and rapid mitochondrial fragmentation in BA, characterized by Drp1 phosphorylation and Opa1 cleavage. Mechanistically, NE‐mediated Drp1 phosphorylation was dependent on Protein Kinase‐A (PKA) activity, whereas Opa1 cleavage required mitochondrial depolarization mediated by FFAs released as a result of lipolysis. This change in mitochondrial architecture was observed both in primary cultures and brown adipose tissue from cold‐exposed mice. Mitochondrial uncoupling induced by NE in brown adipocytes was reduced by inhibition of mitochondrial fission through transient Drp1 DN overexpression. Furthermore, forced mitochondrial fragmentation in BA through Mfn2 knock down increased the capacity of exogenous FFAs to increase energy expenditure. These results suggest that, in addition to its ability to stimulate lipolysis, NE induces energy expenditure in BA by promoting mitochondrial fragmentation. Together these data reveal that adrenergically‐induced changes to mitochondrial dynamics are required for BA thermogenic activation and for the control of energy expenditure.     

Down-regulation of Cyclooxygenase-2 by the Carboxyl Tail of the Angiotensin II Type 1 Receptor

The enzyme cyclooxygenase-2 (COX-2) plays an important role in the kidney by up-regulating the production of the vasoconstrictor hormone angiotensin II (AngII), which in turn down-regulates COX-2 expression via activation of the angiotensin II type 1 receptor (AT₁) receptor. Chemical inhibition of the catalytic activity of COX-2 is a well-established strategy for treating inflammation but little is known of cellular mechanisms that dispose of the protein itself. Here we show that in addition to its indirect negative feedback on COX-2, AT₁ also down-regulates the expression of the COX-2 protein via a pathway that does not involve G-protein or β-arrestin-dependent signaling. Instead, AT₁ enhances the ubiquitination and subsequent degradation of the enzyme in the proteasome through elements in its cytosolic carboxyl tail (CT). We find that a mutant receptor that lacks the last 35 amino acids of its CT (Δ324) is devoid of its ability to reduce COX-2, and that expression of the CT sequence alone is sufficient to down-regulate COX-2. Collectively these results propose a new role for AT₁ in regulating COX-2 expression in a mechanism that deviates from its canonical signaling pathways. Down-regulation of COX-2 by a short peptide that originates from AT₁ may present as a basis for novel therapeutic means of eliminating excess COX-2 protein.     

mTOR complex 1: a key player in neuroadaptations induced by drugs of abuse

The mammalian (or mechanistic) target of rapamycin (mTOR) complex 1 (mTORC1) is a serine and threonine kinase that regulates cell growth, survival, and proliferation. mTORC1 is a master controller of the translation of a subset of mRNAs. In the central nervous system mTORC1 plays a crucial role in mechanisms underlying learning and memory by controlling synaptic protein synthesis. Here, we review recent evidence suggesting that the mTORC1 signaling pathway promotes neuroadaptations following exposure to a diverse group of drugs of abuse including stimulants, cannabinoids, opiates, and alcohol. We further describe potential molecular mechanisms by which drug‐induced mTORC1 activation may alter brain functions. Finally, we propose that mTORC1 is a focal point shared by drugs of abuse to mediate drug‐related behaviors such as reward seeking and excessive drug intake, and offer future directions to decipher the contribution of the kinase to mechanisms underlying addiction.