The Amino-terminal Charge and Core Region Hydrophobicity Interdependently Contribute to the Function of Signal Sequences

Abstract

We have constructed a series of signal sequence mutants that contain negatively charged amino termini and simplified core regions of varying hydrophobicity levels. This series provides a means of exploring the relative roles of the amino terminus and the hydrophobic core region during transport. The signal peptides with highly hydrophobic core regions support a rapid rate of transport in the presence of a negatively charged amino terminus. We have found that these negatively charged mutants are secreted in a manner similar to the wild-type signal sequence; sodium azide and carbonyl cyanide 3-chlorophenylhydrazone treatments indicate that the negatively charged mutants depend on SecA and the protonmotive force, respectively. These same mutants also demonstrate reduced competition with coexpressed beta-lactamase, reflecting the lower overall affinity for the transport pathway due to the net negative charge at the amino terminus. In addition, the pronounced effects of introducing three negative charges support the conclusion that the two regions function in a concerted manner.

Trigger Factor Retards Protein Export in Escherichia coli

“…Although the Bla targeting pathway(s) has never been clearly identified, previous studies have shown that a variety of physiological insults that increase the presence of abnormal proteins (and that likely induce the heat shock response) lead to Bla export defects (7,34). These and other results (5) suggest that Bla is targeted by one or more heat shock-regulated chaperones such as DnaK or GroEL.…”

Identification of Membrane Insertion Sequences of the Rabbit Gastric Cholecystokinin-A Receptor by in VitroTranslation

“…These charges within the sequence of the CCK-A receptor therefore promote membrane insertion, whereas the charges in the N-terminal region of the ␣ chain of the H,K-ATPase present in the HK-M0 vector do not. This result suggests that the positively charged cytoplasmic anchor and the hydrophobic sequence may also have to be in proximity to affect membrane insertion or orientation (35). Furthermore, when the H6 putative transmembrane domain is extended on the C-terminal side, including two positive and one negative charge (HK-M0/CCK6LC), this sequence also promotes a strong glycosylation of the ␤ region, therefore acting as a signal anchor.…”

SecB Dependence of an Exported Protein Is a Continuum Influenced by the Characteristics of the Signal Peptide or Early Mature Region

“…2A). Earlier studies have demonstrated a clear correlation between the hydrophobicity of these signal peptides, the efficiency of in vivo transport (7,15), and cross-linking with P48 and with SecA (28) in vitro and with binding to SecA in reconstituted systems (21,29). secB ϩ and secB null cells transformed with the mutant alkaline phosphatases under the control of the lac promoter were pulse labeled with […”