Glyceraldehyde-3-phosphate dehydrogenase interacts with Rab2 and plays an essential role in endoplasmic reticulum to Golgi transport exclusive of its glycolytic activity

Rab2 requires atypical protein kinase C iota/lambda (aPKC iota/lambda) to promote vesicle formation from vesicular tubular clusters (VTCs). The Rab2-generated vesicles are enriched in recycling proteins suggesting that the carriers are retrograde-directed and retrieve transport machinery back to the endoplasmic reticulum. These vesicles also contained the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We have previously established that GAPDH is required for membrane transport between the endoplasmic reticulum and the Golgi complex. Moreover, GAPDH is phosphorylated by aPKC iota/lambda and binds to the aPKC iota/lambda regulatory domain. In this study, we employed a combination of in vivo and in vitro assays and determined that GAPDH also interacts with Rab2. The site of GAPDH interaction was mapped to Rab2 residues 20-50. In addition to its glycolytic function, GAPDH has multiple intracellular roles. However, the function of GAPDH in the early secretory pathway is unknown. One possibility is that GAPDH ultimately provides energy in the form of ATP. To determine whether GAPDH catalytic activity was critical for transport in the early secretory pathway, a conservative substitution was made at Cys-149 located at the active site, and the mutant was biochemically characterized in a battery of assays. Although GAPDH (C149G) has no catalytic activity, Rab2 recruited the mutant protein to membranes in a quantitative binding assay. GAPDH (C149G) is phosphorylated by aPKC iota/lambda and binds directly to Rab2 when evaluated in an overlay binding assay. Importantly, VSV-G transport between the ER and Golgi complex is restored when an in vitro trafficking assay is performed with GAPDH-depleted cytosol and GAPDH (C149G). These data suggest that GAPDH imparts a unique function necessary for membrane trafficking from VTCs that does not require GAPDH glycolytic activity.     

Role of Ca(2+) in proteolysis-inducing factor (PIF)-induced atrophy of skeletal muscle

Proteolysis-inducing factor (PIF) induces muscle loss in cancer cachexia through a high affinity membrane bound receptor. This study investigates the mechanism by which the PIF receptor communicates to intracellular signalling pathways. C(2)C(12) murine myoblasts were used as a model using PIF purified from MAC16 tumours. Calcium imaging was determined using fura-4-acetoxymethyl ester (Fura-4-AM). PIF induced a rapid rise in Ca(2+)(i), which was completely attenuated by a anti-receptor antibody, or peptides representing 20 mers of the N-terminus of the PIF receptor. Other agents catabolic for skeletal muscle including angiotensin II (AngII) tumour necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) also induced a rise in Ca(2+)(i), but this was not attenuated by anti-PIF-receptor antibody. The rise in Ca(2+)(i) induced by PIF and AngII was completely attenuated by the Zn(2+) chelator D-myo-inositol-1,2,6-triphosphate, and this was reversed by administration of exogenous Zn(2+). The Ca(2+)(i) rise induced by PIF was independent of the presence of extracellular Ca(2+), and attenuated by the Ca(2+) pump inhibitor thapsigargin, suggesting that the Ca(2+)(i) rise was due to release from intracellular stores. This rise in Ca(2+)(i) induced by PIF was attenuated by both the phospholipase C inhibitor U73122 and 2-APB, an inhibitor of the inositol 1,4,5-triphosphate receptor, suggesting the involvement of a G-protein. Binding of the PIF to its receptor in skeletal muscle triggers a rise in Ca(2+)(i), which initiates a signalling cascade leading to a depression in protein synthesis, and an increase in protein degradation.     

Role of β-adrenergic receptors in the anti-obesity and anti-diabetic effects of zinc-α2-glycoprotien (ZAG)

Objectives: The goal of the current study is to determine whether the β-adrenoreceptor (β-AR) plays a role in the anti-obesity and anti-diabetic effects of zinc-α2-glycoprotein (ZAG). Material and methods: This has been investigated in CHO-K1 cells transfected with the human β1-, β2-, β3-AR and in ob/ob mice. Cyclic AMP assays were carried out along with binding studies. Ob/ob mice were treated with ZAG and glucose transportation and insulin were examined in the presence or absence of propranolol. Results: ZAG bound to the β3-AR with higher affinity (Kd 46 ± 1 nM) than the β2-AR (Kd 71 ± 3 nM) while there was no binding to the β1-AR, and this correlated with the increases in cyclic AMP in CHO-K1 cells transfected with the various β-AR and treated with ZAG. Treatment of ob/ob mice with ZAG increased protein expression of β3-AR in gastrocnemius muscle, and in white and brown adipose tissues, but had no effect on expression of β1- and β2-AR. A reduction of body weight was seen and urinary glucose excretion, increase in body temperature, reduction in maximal plasma glucose and insulin levels in the oral glucose tolerance test, and stimulation of glucose transport into skeletal muscle and adipose tissue, were completely attenuated by the non-specific β-AR antagonist propranolol. Conclusion: The results suggest that the effects of ZAG on body weight and insulin sensitivity in ob/ob mice are manifested through a β-3AR, or possibly a β2-AR.     

Techniques for rapid biochemical screening of large numbers of cell clones

Mutant cell lines derived from a variety of organisms can be isolated when a sufficient number of clones are screened. We describe techniques which can be used to clone up to 10⁴ cells/h. The construction of a simple multiple pipet is outlined and its operation in conjunction with multitest culture trays and an efficient replicator is described. The techniques permit screening of any cells able to grow clonally in culture.     

Effect of temperature on seed production in the invasive grass Glyceria maxima (Hartm.) Holmb. (Poaceae) in South Africa

Temperature is one of the main factors that determine sexual reproduction in terrestrial and emergent aquatic plant species. The effect of temperature on sexual reproduction and seed production of Glyceria maxima (Hartm.) Holmb. in the southern hemisphere is unknown. Glyceria maxima collections in February 2010 at three isolated infestations in KwaZulu-Natal failed to yield a single seed, only empty panicles. Laboratory experiments showed that vernalisation had no consistent effect on seed production. Field- and laboratory-grown plants produced seeds in the 2010/2011 season, because of having sufficient time at optimum temperatures required for seed production (1 491 and 1 585 hours, respectively), compared to a shorter period (1 352 hours) of suitable temperatures during the 2009/2010 growing season. An inadequate period of optimum temperatures (15–25°C) during seed production resulted in the lack of seeds in the field in the 2009/2010 growing season. This study showed that temperature and duration of exposure thereto during the seed-production period play vital roles in G. maxima sexual reproduction.