The mechanism of renal glucose transport involves the reabsorption of filtered glucose from the proximal tubule lumen across the brush border membrane (BBM) via a sodium‐dependent transporter, SGLT, and exit across the basolateral membrane via facilitative, GLUT‐mediated, transport. The aim of the present study was to determine the effect of streptozotocin‐induced diabetes on BBM glucose transport. We found that diabetes increased facilitative glucose transport at the BBM by 67.5 % (P < 0.05) – an effect that was abolished by overnight fasting. Western blotting and immunohistochemistry demonstrated GLUT2 expression at the BBM during diabetes, but the protein was undetectable at the BBM of control animals or diabetic animals that had been fasted overnight. Our findings indicate that streptozotocin‐induced diabetes causes the insertion of GLUT2 into the BBM and this may provide a low affinity/high capacity route of entry into proximal tubule cells during hyperglycaemia.

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SummaryVesicles formed by the COPI complex function in retrograde transport from the Golgi to the endoplasmic reticulum (ER). Phosphatidylinositol transfer protein b (PITPb), an essential protein that possesses phosphatidylinositol (PtdIns) and phosphatidylcholine (PtdCho) lipid transfer activity is known to localise to the Golgi and ER but its role in these membrane systems is not clear. To examine the function of PITPb at the Golgi-ER interface, RNA interference (RNAi) was used to knockdown PITPb protein expression in HeLa cells. Depletion of PITPb leads to a decrease in PtdIns(4)P levels, compaction of the Golgi complex and protection from brefeldin-Amediated dispersal to the ER. Using specific transport assays, we show that anterograde traffic is unaffected but that KDEL-receptordependent retrograde traffic is inhibited. This phenotype can be rescued by expression of wild-type PITPb but not by mutants defective in docking, PtdIns transfer and PtdCho transfer. These data demonstrate that the PtdIns and PtdCho exchange activity of PITPb is essential for COPI-mediated retrograde transport from the Golgi to the ER.

Of many lipid transfer proteins identified, all have been implicated in essential cellular processes, but the activity of none has been demonstrated in intact cells. Among these, phosphatidylinositol transfer proteins (PITP) are of particular interest as they can bind to and transfer phosphatidylinositol (PtdIns) – the precursor of important signalling molecules, phosphoinositides – and because they have essential functions in neuronal development (PITPα) and cytokinesis (PITPβ). Structural analysis indicates that, in the cytosol, PITPs are in a ‘closed’ conformation completely shielding the lipid within them. But during lipid exchange at the membrane, they must transiently ‘open’. To study PITP dynamics in intact cells, we chemically targeted their C95 residue that, although non-essential for lipid transfer, is buried within the phospholipid-binding cavity, and so, its chemical modification prevents PtdIns binding because of steric hindrance. This treatment resulted in entrapment of open conformation PITPs at the membrane and inactivation of the cytosolic pool of PITPs within few minutes. PITP isoforms were differentially inactivated with the dynamics of PITPβ faster than PITPα. We identify two tryptophan residues essential for membrane docking of PITPs.

CD8 glycoproteins are expressed as either αα homodimers or αβ heterodimers on the surface of T cells. CD8αβ is a more efficient coreceptor than the CD8αα for peptide Ag recognition by TCR. Each CD8 subunit is composed of four structural domains, namely, Ig-like domain, stalk region, transmembrane region, and cytoplasmic domain. In an attempt to understand why CD8αβ is a better coreceptor than CD8αα, we engineered, expressed, and functionally tested a chimeric CD8α protein whose stalk region is replaced with that of CD8β. We found that the β stalk region enhances the coreceptor function of chimeric CD8αα to a level similar to that of CD8αβ. Surprisingly, the β stalk region also restored functional activity to an inactive CD8α variant, carrying an Ala mutation at Arg8 (R8A), to a level similar to that of wild-type CD8αβ. Using the R8A variant of CD8α, a panel of anti-CD8α Abs, and three MHC class I (MHCI) variants differing in key residues known to be involved in CD8α interaction, we show that the introduction of the CD8β stalk leads to a different topology of the CD8α-MHCI complex without altering the overall structure of the Ig-like domain of CD8α or causing the MHCI to employ different residues to interact with the CD8α Ig domain. Our results show that the stalk region of CD8β is capable of fine-tuning the coreceptor function of CD8 proteins as a coreceptor, possibly due to its distinct protein structure, smaller physical size and the unique glycan adducts associated with this region.