The Cotranslational Maturation Program for the Type II Membrane Glycoprotein Influenza Neuraminidase

Abstract

The earliest steps in nascent protein maturation greatly affect its overall efficiency. Constraints placed on maturing proteins at these early stages limit available conformations and help to direct the native maturation process. For type II membrane proteins, these cotranslational constraints include N-and C-terminal membrane tethering, chaperone binding, and disulfide bond formation. The cotranslational maturation process for the type II membrane glycoprotein influenza neuraminidase (NA) was investigated to provide a deeper understanding of these initial endoplasmic reticulum events. The type II orientation provides experimental advantages to monitor the first maturation steps. Calnexin was shown to cotranslationally interact with NA prior to calreticulin. These interactions were required for the efficient maturation of NA as it prematurely formed intramolecular disulfides and aggregated when calnexin and calreticulin interactions were abolished. Lectin chaperone binding slowed the NA maturation process, increasing its fidelity. Carbohydrates were required for NA maturation in a regiospecific manner. A subset of NA formed intermolecular disulfides and oligomerized cotranslationally. This fraction increased in the absence of calnexin and calreticulin binding. NA dimerization also occurred for an NA mutant lacking the critical large loop disulfide bond, indicating that dimerization did not require proper NA oxidation. The strict evaluation of proper maturation carried out by the quality control machinery was instilled at the tetramerization step. This study illustrates the type II membrane protein maturation process and shows how important cotranslational events contribute to the proper cellular maturation of glycoproteins.

Assembly of Subtype 1 Influenza Neuraminidase Is Driven by Both the Transmembrane and Head Domains

“…Previous work demonstrated that NA rapidly dimerizes within ϳ2.5 min and this can occur co-translationally (25,27). Additionally, conformation specific antibodies revealed a unique time lag of ϳ5 min between NA oligomerization and when NA reached its native tetrameric conformation (25).…”

“…NA oligomerization occurs rapidly and efficiently within the endoplasmic reticulum, possibly through an N-terminal directed process (25)(26)(27). However, the direct role of the TMD in NA assembly has received little attention.…”

Sorting things out through endoplasmic reticulum quality control

“…As small proteins may be able to fold without the assistance of chaperones, it would be of interest to determine if glucosidase II trimming can be controlled to by-pass lectin chaperone binding so that these chaperones can focus their attention on more complex proteins that require their assistance for proper maturation. The number and location of glycans appears to dictate to some extent the molecular choreography or the chaperone binding profile for the individual chaperones (Hebert et al, 1997, Molinari and Helenius, 2000, Wang et al, 2005, Wang et al, 2008). While non-glycosylated proteins or regions may be aided by the Hsp70 chaperone BiP and protein disulfide isomerase family members such as PDI, glycans provide the dominant chaperone signal.…”

Protein Folding in the Endoplasmic Reticulum

“…The necessity of a modification at a specific site is highly protein and site dependent. Whereas some sites of modification are absolutely required for efficient maturation, others are completely dispensable (Hebert et al 1997;Wang et al 2008). Sometimes the location is not critical but the total number of glycans is.…”