Domain Organization of Long Signal Peptides of Single-Pass Integral Membrane Proteins Reveals Multiple Functional Capacity

Abstract

Targeting signals direct proteins to their extra - or intracellular destination such as the plasma membrane or cellular organelles. Here we investigated the structure and function of exceptionally long signal peptides encompassing at least 40 amino acid residues. We discovered a two-domain organization (“NtraC model”) in many long signals from vertebrate precursor proteins. Accordingly, long signal peptides may contain an N-terminal domain (N-domain) and a C-terminal domain (C-domain) with different signal or targeting capabilities, separable by a presumably turn-rich transition area (tra). Individual domain functions were probed by cellular targeting experiments with fusion proteins containing parts of the long signal peptide of human membrane protein shrew-1 and secreted alkaline phosphatase as a reporter protein. As predicted, the N-domain of the fusion protein alone was shown to act as a mitochondrial targeting signal, whereas the C-domain alone functions as an export signal. Selective disruption of the transition area in the signal peptide impairs the export efficiency of the reporter protein. Altogether, the results of cellular targeting studies provide a proof-of-principle for our NtraC model and highlight the particular functional importance of the predicted transition area, which critically affects the rate of protein export. In conclusion, the NtraC approach enables the systematic detection and prediction of cryptic targeting signals present in one coherent sequence, and provides a structurally motivated basis for decoding the functional complexity of long protein targeting signals.

Adherens junctional associated protein-1: A novel 1p36 tumor suppressor candidate in gliomas

“…2) (42). The relatively long signal peptide also has a role in the cellular location of AJAP1 (43,44). The signal peptide of AJAP1 conforms to the NtraC model describing the organization of long signal peptides, i.e., an N-terminal subdomain, a β-turn-rich transition area, and a C-terminal subdomain.…”

“…The N-domain alone directs AJAP1 primarily to mitochondria. This cryptic mitochondrial targeting signal has been speculated to be active only under certain physiological conditions, e.g., apoptosis (43,44). Further analysis of the AJAP1 protein sequence revealed a putative nuclear localization signal in the predicted extracellular domain and putative glycosylation signals in the cytoplasmic domain (35).…”

Type V Secretion Systems in Bacteria

“…Generally, the N-terminal signal sequences in proteins show a tripartite organization of n, h, c, referring to the regions of N-terminal, hydrophobic, and cleavage site (46). Several studies showed variations (n1-h1-n2-h2; n2-h2) of the signal peptide tripartite organization in type V systems (4,47) and called the region the extended signal peptide region (ESPR), which might have additional functions besides targeting or protein translocation (48)(49)(50). It is important to note that using simple sequence analysis tools such as BLAST and ClustalX, Desvaux and his colleagues showed that the ESPR sequence pattern is phylogenetically restricted among the Gram-negative bacterial type V-secreted proteins from the classes of βand γ-Proteobacteria (47).…”

Alternative exon usage creates novel transcript variants of tumor suppressor SHREW-1 gene with differential tissue expression profile

“…Both novel transcripts arise by alternative exon usage. The originally identified shrew-1 transcript encodes a protein of 411 aa (isoform 1) with an unusually long signal peptide (SP, aa 1-43) that contains functional distinct targeting subdomains (Bharti et al, 2004; Hiss et al, 2008). The first newly identified shrew-1 transcript variant (referred to as isoform 2) starts with a novel exon (E1a), leading to an 11 aa shorter SP, that might bring so far unknown subcellular targeting features to shrew-1.…”