Membrane Proteins In Mammalian Cells Research Articles

Immunolocalization of the intracellular sites of accumulation of mutant influenza hemagglutinins by Electron Microscopy

The hemagglutinin (HA) of influenza virus has been used as a model system to study the biosynthesis and intracellular transport of integral membrane proteins in mammalian cells. To investigate the role of protein structure in facilitating transport along the secretory pathway, we have examined the expression in monkey CV-1 cells of a large number of mutant HA molecules. The majority of the HA mutants do not progress along the secretory pathway and accumulate in the endoplasmic reticulum (ER), and we have shown that assembly of newly-synthesized HA monomers into correctly folded trimeric structures is required for transport of the protein to the Golgi apparatus. By contrast, only one HA mutant has beegn characterized whose transport is blocked at a post-Golgi stage of the pathway and thus little is known about the factors involved in the sorting of the HA molecule from the Golgi apparatus to the plasma membrane (PM). In this study we are using electron microscopy to precisely define the intracellular site of accumulation of two mutant HAs whose transport is blocked at different stages of the secretory pathway. In mutant HAJS67, a cysteine residue (cys67) involved in a key disulfide bond has been substituted by a serine residue. In mutant HA164, the 10 amino acid cytoplasmic tail of the wild-type HA has been replaced by a non-homologous sequence of 16 amino acids. Biochemical and immunof1uoresence analyses have indicated that HAJS67 molecules remain in the ER compartment while HA164 is largely confined to a post-Golgi compartment, possibly the trans Golgi network (TGN).

Intracellular transport of a variant surface glycoprotein in Trypanosoma brucei.

Trypanosome variant surface glycoproteins (VSGs) have a novel glycan-phosphatidylinositol membrane anchor, which is cleavable by a phosphatidylinositol-specific phospholipase C. A similar structure serves to anchor some membrane proteins in mammalian cells. Using kinetic and ultrastructural approaches, we have addressed the question of whether this structure directs the protein to the cell surface by a different pathway from the classical one described in other cell types for plasma membrane and secreted glycoproteins. By immunogold labeling on thin cryosections we were able to show that, intracellularly, VSG is associated with the rough endoplasmic reticulum, all Golgi cisternae, and tubulovesicular elements and flattened cisternae, which form a network in the area adjacent to the trans side of the Golgi apparatus. Our data suggest that, although the glycan-phosphatidylinositol anchor is added in the endoplasmic reticulum, VSG is nevertheless subsequently transported along the classical intracellular route for glycoproteins, and is delivered to the flagellar pocket, where it is integrated into the surface coat. Treatment of trypanosomes with 1 microM monensin had no effect on VSG transport, although dilation of the trans-Golgi stacks and lysosomes occurred immediately. Incubation of trypanosomes at 20 degrees C, a treatment that arrests intracellular transport from the trans-Golgi region to the cell surface in mammalian cells, caused the accumulation of VSG molecules in structures of the trans-Golgi network, and retarded the incorporation of newly synthesized VSG into the surface coat.