Plasma cells daily secrete their own mass in antibodies, which fold and assemble in the endoplasmic reticulum (ER). To reach these levels, cells require pERp1, a novel lymphocyte-specific small ER-resident protein, which attains expression levels as high as BiP when B cells differentiate into plasma cells. Although pERp1 has no homology with known ER proteins, it does contain a CXXC motif typical for oxidoreductases. In steady state, the CXXC cysteines are locked by two parallel disulfide bonds with a downstream C(X)6C motif, and pERp1 displays only modest oxidoreductase activity. pERp1 emerged as a dedicated folding factor for IgM, associating with both heavy and light chains and promoting assembly and secretion of mature IgM.

The family of low density lipoprotein (LDL) receptors mediate uptake of a plethora of ligands from the circulation and couple this to signaling, thereby performing a crucial role in physiological processes including embryonic development, cancer development, homeostasis of lipoproteins, viral infection, and neuronal plasticity. Structural integrity of individual ectodomain modules in these receptors depends on calcium, and we showed before that the LDL receptor folds its modules late after synthesis via intermediates with abundant non-native disulfide bonds and structure. Using a radioactive pulsechase approach, we here show that for proper LDL receptor folding, calcium had to be present from the very early start of folding, which suggests at least some native, essential coordination of calcium ions at the still largely non-native folding phase. As long as the protein was in the endoplasmic reticulum (ER), its folding was reversible, which changed only upon both proper incorporation of calcium and exit from the ER. Coevolution of protein folding with the high calcium concentration in the ER may be the basis for the need for this cation throughout the folding process even though calcium is only stably integrated in native repeats at a later stage.

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Ih is a poorly selective cation current that activates upon hyperpolarization, present in various types of neurons. Our aim was to perform a detailed thermodynamic analysis of Ih gating kinetics, in order to assess putative structural changes associated with its activation and deactivation. To select dorsal root ganglia neurons that exhibit large Ih, we applied a current signature method by Petruska et al. (J Neurophysiol 84:2365-2379, 2000) and found appropriate neurons in cluster 4. Currents elicited by 3,000-ms hyperpolarizing pulses at 25 and 33 degrees C were fitted with double exponential functions, yielding time constants similar to those of HCN1. The fast activation and deactivation rates showed temperature coefficients (Q10) of 2.9 and 3.1, respectively, while Q10 of the absolute conductance was 1.3. Using the Arrhenius-Eyring formalism we computed heights of voltage-independent Gibbs free energy and entropy barriers for each rate. The free energy barriers of the fast rates were just approximately 2RT units lower than those of the corresponding slow rates (31.3 vs. 33.2RT for activation, and 24.7 vs. 25.8RT for deactivation, at 25 degrees C). Interestingly, the entropy barriers of the slow rates were negative: -15.2R units for activation and -11.9R units for deactivation, compared to 4.6 and 1.3R units, respectively, for the fast component. The equivalent gating charge (zg) (3.75 +/- 0.32, mean +/- SEM, at 25 degrees C) and half-activation potential (V1/2) (-70.0 +/- 1.3 mV at 25 degrees C) did not vary significantly with temperature.

The Canopy (CNPY) family consists of four members predicted to be soluble proteins localized to the endoplasmic reticulum (ER). They are involved in a wide array of processes, including angiogenesis, cell adhesion, and host defense. CNPYs are thought to do so via regulation of secretory transport of a diverse group of proteins, such as immunoglobulin M, growth factor receptors, toll‐like receptors, and the low‐density lipoprotein receptor. Thus far, a comparative analysis of the mammalian CNPY family is missing. Bioinformatic analysis shows that mammalian CNPYs, except the CNPY1 homolog, have N‐terminal signal sequences and C‐terminal ER‐retention signals and that mammals have an additional member CNPY5, also known as plasma cell‐induced ER protein 1/marginal zone B cell‐specific protein 1. Canopy proteins are particularly homologous in four hydrophobic alpha‐helical regions and contain three conserved disulfide bonds. This sequence signature is characteristic for the saposin‐like superfamily and strongly argues that CNPYs share this common saposin fold. We showed that CNPY2, 3, 4, and 5 (termed CNPYs) localize to the ER. In radioactive pulse‐chase experiments, we found that CNPYs rapidly form disulfide bonds and fold within minutes into their native forms. Disulfide bonds in native CNPYs remain sensitive to low concentrations of dithiothreitol (DTT) suggesting that the cysteine residues forming them are relatively accessible to solutes. Possible roles of CNPYs in the folding of secretory proteins in the ER are discussed.