Research

Gene expression: components and mechanisms of intracellular protein transport and protein folding

Transport into the endoplasmic reticulum (ER) is the first biosynthetic step for secretory proteins. This highly regulated process is critical for the biosynthesis of many membrane proteins as well, and requires cytosolic and ER proteins, many of which were first identified using yeast genetics. The Zimmermann group studies the protein translocase that mediates the cotranslational insertion of proteins into the membrane of the pancreatic endoplasmic reticulum. Biochemical, biophysical, structural, and cell biological approaches are combined in order to elucidate the mechanism and the regulation of components of this protein translocase. The products of SIL1, SEC62, and SEC63 genes act in concert with the Sec61 complex and the molecular chaperones BiP and Grp170 to transport proteins into the ER. A special emphasis is placed on transport components of the ER-membrane that associate with the tunnel exit of cytosolic ribosomes (such as Sec61 complex, ERj1, Sec62). Interestingly, recent  genetic work has linked mutations in the human and murine SIL1 genes to neurodegeneration and mutations in the human SEC63 gene to autosomal dominant polycystic liver disease. Furthermore, mutations in the SEC63 gene and overexpresion of the SEC62 gene are associated with variious human cancers. This last aspect is the focus of collaboration between the Zimmermann group and the clinic of urology.

The ER also plays a central role in cellular calcium homeostasis, which is essential for cellular Ca²⁺ signaling. In the resting cell, the cytosolic Ca²⁺ concentration is low due to the action of plasma membrane resident pumps and exchangers (50-100 nM). Simultaneously, the Ca²⁺ concentration in the ER lumen is high due to the action of sarcoplasmic endoplasmic reticulum calcium ATPases or SERCA (100-800 µM). This steep intracellular Ca²⁺ gradient is essential for many signal transduction pathways, but constantly challenged by passive Ca²⁺ efflux from the ER or Ca²⁺ leakage. Indirect evidence from various laboratories has first suggested that the ER membrane resident Sec61 complex, which facilitates membrane insertion and translocation of nascent and newly-synthesized precursor polypeptides into the ER, may transiently contribute to the ER Ca²⁺ leak after completion of protein translocation. Tthis concept was confirmed by our observations that the open Sec61 complex is indeed Ca²⁺ permeable and that silencing of the SEC61A1 gene in HeLa cells prevents the Ca²⁺ leakage that is linked to completion of protein translocation. Furthermore, we demonstrated by the combination of siRNA-mediated gene silencing and live cell Ca²⁺ imaging in cytosol as well as ER lumen that the ER lumenal Hsp70-type chaperone BiP or Grp78 and its co-chaperones are involved in gating of the Sec61 complex to the non-conducting state and identified the crucial binding site for BiP on ER lumenal Loop 7 of the Sec61a subunit. Once Ca²⁺ has started to leak out, cytosolic Ca²⁺-calmodulin was shown to contribute to Sec61 channel closing, which involves the ER membrane resident EF-hand protein Sec62, and a crucial IQ-motif in the cytosolic N-terminus of Sec61a as binding site for Ca²⁺-calmodulin. These latter observations culminated in the proposal of a novel therapeutic strategy for the treatment of SEC62 over-expressing tumors, such as prostateThe ER also plays a central role in cellular calcium homeostasis, which is essential for cellular Ca²⁺ signaling. In the resting cell, the cytosolic Ca²⁺ concentration is low due to the action of plasma membrane resident pumps and exchangers (50-100 nM). Simultaneously, the Ca²⁺ concentration in the ER lumen is high due to the action of sarcoplasmic endoplasmic reticulum calcium ATPases or SERCA (100-800 µM). This steep intracellular Ca²⁺ gradient is essential for many signal transduction pathways, but constantly challenged by passive Ca²⁺ efflux from the ER or Ca²⁺ leakage. Indirect evidence from various laboratories has first suggested that the ER membrane resident Sec61 complex, which facilitates membrane insertion and translocation of nascent and newly-synthesized precursor polypeptides into the ER, may transiently contribute to the ER Ca²⁺ leak after completion of protein translocation. Tthis concept was confirmed by our observations that the open Sec61 complex is indeed Ca²⁺ permeable and that silencing of the SEC61A1 gene in HeLa cells prevents the Ca²⁺ leakage that is linked to completion of protein translocation. Furthermore, we demonstrated by the combination of siRNA-mediated gene silencing and live cell Ca²⁺ imaging in cytosol as well as ER lumen that the ER lumenal Hsp70-type chaperone BiP or Grp78 and its co-chaperones are involved in gating of the Sec61 complex to the non-conducting state and identified the crucial binding site for BiP on ER lumenal Loop 7 of the Sec61a subunit. Once Ca2+ has started to leak out, cytosolic Ca²⁺-calmodulin was shown to contribute to Sec61 channel closing, which involves the ER membrane resident EF-hand protein Sec62, and a crucial IQ-motif in the cytosolic N-terminus of Sec61a as binding site for Ca²⁺-calmodulin. These latter observations culminated in the proposal of a novel therapeutic strategy for the treatment of SEC62 over-expressing tumors, such as prostate or lung cancer and a patent application.