N-Glycosylation is a cotranslational and post-translational process of proteins that may influence protein folding, maturation, stability, trafficking, and consequently cell surface expression of functional channels. Here we have characterized two consensus N-glycosylation sequences of a voltage-gated K+ channel (Kv3.1). Glycosylation of Kv3.1 protein from rat brain and infected Sf9 cells was demonstrated by an electrophoretic mobility shift assay. Digestion of total brain membranes with peptide N glycosidase F (PNGase F) produced a much faster-migrating Kv3.1 immunoband than that of undigested brain membranes. To demonstrate N-glycosylation of wild-type Kv3.1 in Sf9 cells, cells were treated with tunicamycin. Also, partially purified proteins were digested with either PNGase F or endoglycosidase H. Attachment of simple-type oligosaccharides at positions 220 and 229 was directly shown by single (N229Q and N220Q) and double (N220Q/N229Q) Kv3.1 mutants. Functional measurements and membrane fractionation of infected Sf9 cells showed that unglycosylated Kv3.1s were transported to the plasma membrane. Unitary conductance of N220Q/N229Q was similar to that of the wild-type Kv3.1. However, whole cell currents of N220Q/N229Q channels had slower activation rates, and a slight positive shift in voltage dependence compared to wild-type Kv3.1. The voltage dependence of channel activation for N229Q and N220Q was much like that for N220Q/N229Q. These results demonstrate that the S1-S2 linker is topologically extracellular, and that N-glycosylation influences the opening of the voltage-dependent gate of Kv3.1. We suggest that occupancy of the sites is critical for folding and maturation of the functional Kv3.1 at the cell surface.

The minimal sequence requirement for a peroxisome-targeting signal was investigated using an in vitro import system. Carboxyl-terminal sequences Ser-Lys-Leu (SKL) and Leu-Gln-Ser-Lys-Leu (LQSKL) of acyl-CoA oxidase (AOX) directed to peroxisomes the fused proteins with import-incompetent forms of AOX and catalase that had been truncated, implying that the SKL tripeptide functions as a targeting signal. Elimination of the entire SKL sequence or deletion of any 1 or 2 amino acids in the sequence abolished the import activity of AOX. Substitution of alanine for serine did not affect the import activity. Topogenic activity was retained when lysine was mutated to either arginine or histidine, whereas mutation to glutamic acid completely abolished the activity. A synthetic peptide comprising the carboxyl-terminal 10 amino acid residues of AOX inhibited the import of the authentic AOX polypeptide, whereas other peptides in which SKL was mutated, deleted, or internally located were not effective. The uptake of AOX was little affected by the peptide with an amidated alpha-carboxyl group. These results strongly suggest that the carboxyl-terminal SKL motif sequence (Ser/Ala)-(Lys/Arg/His)-Leu functions as a topogenic signal in translocation of proteins into peroxisomes, requiring the whole tripeptide sequence with a free alpha-COOH group at the carboxyl terminus.