Recombinant proteins containing tetracysteine tags can be successively labeled in living cells with different colors of biarsenical fluorophores so that older and younger protein molecules can be sharply distinguished by both fluorescence and electron microscopy. Here we used this approach to show that newly synthesized connexin43 was transported predominantly in 100- to 150-nanometer vesicles to the plasma membrane and incorporated at the periphery of existing gap junctions, whereas older connexins were removed from the center of the plaques into pleiomorphic vesicles of widely varying sizes. Selective imaging by correlated optical and electron microscopy of protein molecules of known ages will clarify fundamental processes of protein trafficking in situ.

Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments1 or require light and are difficult to use2. Here we report the development of a simple and robust EM genetic tag, called “APEX,” that is active in all cellular compartments and does not require light. APEX is a monomeric 28 kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins. We also fused APEX to the N- or C-terminus of the mitochondrial calcium uniporter (MCU), a newly identified channel whose topology is disputed3,4. MCU-APEX and APEX-MCU give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N-and C-termini of MCU face the matrix.

Pannexins are newly discovered channel proteins expressed in many different tissues and abundantly in the vertebrate central nervous system. Based on membrane topology, folding and secondary structure prediction, pannexins are proposed to form gap junction-like structures. We show here that Pannexin1 forms a hexameric channel and reaches the cell surface but, unlike connexins, is N-glycosylated. Using site-directed mutagenesis we analyzed three putative N-linked glycosylation sites and examined the effects of each mutation on channel expression. We show for the first time that Pannexin1 is glycosylated at Asn-254 and that this residue is important for plasma membrane targeting. The glycosylation of Pannexin1 at its extracellular surface makes it unlikely that two oligomers could dock to form an intercellular channel. Ultrastructural analysis by electron microscopy confirmed that Pannexin1 junctional areas do not appear as canonical gap junctions. Rather, Pannexin1 channels are distributed throughout the plasma membrane. We propose that N-glycosylation of Pannexin1 could be a significant mechanism for regulating the trafficking of these membrane proteins to the cell surface in different tissues.Intercellular communication takes place in two different ways: release of molecules such as neurotransmitters, hormones, or ATP; and formation of intercellular channels that connect directly the cytoplasm of adjacent cells. Multicellular organisms have evolved to use different types of intercellular channels, called gap junctions. In vertebrates, a gap junction channel is formed by the apposition of two hexameric assemblies of connexins (Cx), 2 one from each adjacent cell. A new class of proteins analogous to connexins in their folding, but completely unrelated in sequence has been identified in invertebrates. These proteins were named innexins (invertebrate connexin analogs, abbreviated as Inx). Despite the conserved structure, properties, and functions of gap junctions in vertebrates and invertebrates, connexin and innexin structures evolved independently.Mammalian analogs of innexins were recently identified in the human genome sequence and the term pannexins (from the Greek word pan, "throughout " and the Latin nexus, "connection, " abbreviated as Panx) was coined to name this new class of proteins (1). Pannexins have some sequence similarity to invertebrate innexin proteins, but share no homology with vertebrate connexins. It has been speculated that there is an evolutionary relationship between pannexins and innexins. Both groups of proteins are predicted to have four transmembrane regions, two extracellular loops and intracellular amino (NH 2 ) and carboxyl (COOH) termini. The human pannexin family consists of three members: Pannexin1 (Panx1, 426 amino acids, 47.6 kDa), Pannexin2 (Panx2, 664 amino acids, 73.3 kDa), and Pannexin3 (Panx3, 392 amino acids, 44.7 kDa). Pannexins are expressed in many different systems and abundantly in the vertebrate central nervous system. Based on membrane topology, folding and seconda...

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