MBB 430 - Lecture 2

Who discovered the secretory pathway?

George Palade

What is the function of pancreatic acinar cells?

Secrete digestive enzymes via zymogen granules

What is the starting point of the secretory pathway?

Rough Endoplasmic Reticulum (RER)

What labeling method was used to study protein transport?

Pulse-chase labeling

What organelle do secretory proteins enter after the ER?

Golgi apparatus

What are zymogen granules?

Storage vesicles for digestive enzymes in acinar cells

What is the purpose of fractionating zymogen granules?

To verify purity for EM studies

How does 14C-Leu labeling behave in the ER after a chase?

It drops immediately

When does 14C-Leu accumulate in zymogen granules?

37 minutes after chase

What does light microscopy show in pulse-chase experiments?

Protein transit from ER to lumen via Golgi and granules

What does EM add to pulse-chase analysis?

Higher resolution and quantification of labeled organelles

At what time does label appear in Golgi-associated vesicles?

3 to 7 minutes post-chase

When do zymogen granules begin to show label?

Around 37 minutes post-chase

Why was there a discrepancy between fractionation and EM labeling?

Contamination of zymogen granules in fraction

What conclusion did Palade draw from P/C studies?

Secretion starts in the ER

Do secretory proteins travel freely in the cytoplasm?

No, they are transported in vesicles

How are secretory proteins moved from ER to Golgi?

Via small Golgi vesicles

Who pioneered the in vitro reconstitution of secretion?

Jim Rothman

What hybrid approach did Rothman use?

Mutant cell fractions complemented with wild-type in vitro

What virus protein was used in Golgi transport assay?

VSV-G (vesicular stomatitis virus glycoprotein)

What was the donor cell line used in the assay?

15B mutant CHO cells

What defect is present in 15B CHO cells?

Lack UDP-GlcNAc transferase

How is transport measured in the assay?

3H-GlcNAc addition to VSV-G in acceptor cells

What technique isolates labeled G protein?

Immunoprecipitation

What proves the donor fraction is Golgi?

Presence of Golgi enzymes like mannosidase I

What proves the acceptor fraction is Golgi?

Also contains Golgi enzymes

What protects transferred G protein from protease?

Sealed compartments

How soon after CHX addition does transport occur?

Within 10 minutes

What does CHX inhibit?

Protein synthesis

What does more cytosol do to in vitro transport?

Increases rate and extent of transport

What components are needed for in vitro transport?

15B donor, WT acceptor, cytosol, ATP

What cytosolic factors are required for transport?

NSF, SNAPs, SNAREs

What motif targets proteins to ER?

N-terminal signal peptide

What motif targets proteins to mitochondria?

N-terminal presequence

What motif targets proteins to chloroplasts?

N-terminal transit peptide

What motif targets proteins to peroxisomes?

C-terminal SKL (PTS1) or internal PTS2

What motif targets proteins to the nucleus?

Internal NLS

What motif targets proteins to lysosomes?

Mannose-6-phosphate

What motif enables ER retrieval?

C-terminal HDEL (yeast) or KDEL

What award did George Palade win for his work?

Nobel Prize 1974