A—Topogenesis of membrane proteins at the Sec61 translocon

In recent years, the structure of the Sec61/SecY translocon, the major gateway for secretion and membrane

protein integration, has been determined in detail. Yet, the mechanism and dynamics of signal sequence

insertion and transmembrane integration remain challenging questions. Systematic analysis with marginally

hydrophobic sequences suggested membrane integration to be the result of thermodynamic equilibration

between the pore and the membrane. Our lab has contributed to uncover principles of signal orientation and

how the translocon defines the hydrophobicity threshold for membrane integration. Now, our goals are to

analyze the dynamic aspects of topogenesis by studying ...

• how sequences downstream of a transmembrane domain significantly regulate its membrane integration

• how BiP as a driving force of protein translocation affects polypeptide integration vs. translocation

• how the interaction of charged residues in transmembrane domains affects the energetics of integration

• the requirements for reinsertion of Ncyt/Cexo transmembrane domains in multispanning proteins

• how decatransin inhibits signal insertion, potentially as a signal sequence mimick.

Our experimental approach is to challenge the translocon in vivo in mammalian cells and in yeast with

model substrates and/or to specifically mutagenize the translocon in yeast.

B—Intracellular protein sorting

Protein transport in the late secretory pathway from the trans-Golgi network (TGN) to the cell surface is still

poorly characterized, also because there are parallel, compensating pathways. Known transport components

at the TGN are clathrin and its adaptor complex AP-1, as well as AP-3 and AP-4 (with or without clathrin).

We plan to analyze TGN sorting mechanisms and pathways by determining the transport kinetics in pulsechase

experiments with [35S]sulfate labeling at the TGN. Endosomal ablation will be used to detect indirect

pathways via endosomes. To analyze the role of candidate machinery, we will primarily employ the

'knocksideways' approach, which allows to efficiently deplete a tagged protein from the cytosol within a few

minutes, without the risk of adaptation or indirect effects as in other silencing approaches. We are initially

producing knocksideways cells for clathrin, AP-1, AP-3, and Arf1 to analyze the effects of their acute

inactivation on TGN exit kinetics. We will study and compare different classes of proteins including

transmembrane, GPI-anchored, soluble, proteoglycan, and lysosomal proteins. Where possible, we will back

up our findings by mutagenesis of potential sorting motifs.

Sorting of peptide hormones into secretory granules at the TGN was proposed to correspond to formation of

functional amyloids. For provasopressin, we found that the hormone nonapeptide and the glycopeptide are

responsible for fibrillar aggregation of diabetes insipidus mutants in the endoplasmic reticulum, but also for

granule sorting of the wild-type protein. Vasopressin consists of a conspicuous disulfide loop of 6 residues.

Many other prohormones contain short disulfide loops of 5–12 residues. We will test the hypothesis that this

structure acts as a general device for amyloid-like aggregation and analyze the properties of these structures

in fibrillar aggregation and granule sorting.

In addition, we have studied Rabaptin5, a potential connector between AP-1/clathrin and endosomal Rab

proteins and identified FIP200, a component of the Atg1/ULK autophagy complex, as a new Rabaptin5

interactor. We plan to characterize the role of Rabaptin5 as a regulator of endosomal autophagy.